Response regulator

ABSTRACT

The invention provides response regulator polypeptides and polynucleotides encoding response regulator polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are methods for utilizing response regulator polypeptides to screen for antibacterial compounds.

RELATED APPLICATIONS

This application claims benefit of Provisional Patent Application Ser.No. 60/050,332, filed Jun. 20, 1997.

FIELD OF THE INVENTION

This invention relates to newly identified polynucleotides andpolypeptides, and their production and uses, as well as their variants,agonists and antagonists, and their uses. In particular, the inventionrelates to polynucleotides and polypeptides of the response regulatorfamily, as well as their variants, hereinafter referred to as "responseregulator," "response regulator polynucleotide(s)," and "responseregulator polypeptide(s)" as the case may be

BACKGROUND OF THE INVENTION

The Streptococci make up a medically important genera of microbes knownto cause several types of disease in humans, including, for example,otitis media, conjunctivitis, pneumonia, bacteremia, meningitis,sinusitis, pleural empyema and endocarditis, and most particularlymeningitis, such as for example infection of cerebrospinal fluid. Sinceits isolation more than 100 years ago, Streptococcus pneumoniae has beenone of the more intensively studied microbes. For example, much of ourearly understanding that DNA is, in fact, the genetic material waspredicated on the work of Griffith and of Avery, Macleod and McCartyusing this microbe. Despite the vast amount of research with S.pneumoniae, many questions concerning the virulence of this microberemain. It is particularly preferred to employ Streptococcal genes andgene products as targets for the development of antibiotics.

The frequency of Streptococcus pneumoniae infections has risendramatically in the past few decades. This has been attributed to theemergence of multiply antibiotic resistant strains and an increasingpopulation of people with weakened immune systems. It is no longeruncommon to isolate Streptococcus pneumoniae strains which are resistantto some or all of the standard antibiotics. This phenomenon has createdan unmet medical need and demand for new anti-microbial agents,vaccines, drug screening methods, and diagnostic tests for thisorganism.

Moreover, the drug discovery process is currently undergoing afundamental revolution as it embraces "functional genorics," that is,high throughput genome- or gene-based biology. This approach is rapidlysuperseding earlier approaches based on "positional cloning" and othermethods. Functional genomics relies heavily on the various tools ofbioinformatics to identify gene sequences of potential interest from themany molecular biology databases now available as well as from othersources. There is a continuing and significant need to identify andcharacterize further genes and other polynucleotides sequences and theirrelated polypeptides, as targets for drug discovery.

Many two component signal transduction systems (TCSTS) have beenidentified in bacteria (Stock, J. B., Ninfa, A. J. & Stock, A. M.(1989)Microbiol. Rev. 53, 450-490). These are involved in the bacterium'sability to monitor its surroundings and adapt to changes in itsenvironment. Several of these bacterial TCSTS are involved in virulenceand bacterial pathogenesis within the host.

Response regulators are components of the TCSTS. These proteins arephosphorylated by histidine kinases and in turn once phosphorylatedeffect the response, often through a DNA binding domain becomingactivated. The response regulators are characterized by a conservedN-terminal domain of approximately 100 amino acids. The N-terminaldomains of response regulators as well as retaining five functionallyimportant residues, corresponding to the residues D12, D13, D57, T87,K109 in CheY (Matsumura, P., Rydel, J. J., Linzmeier, R. & Vacante, D.(1984) J. Bacteriol. 160, 36-41), have conserved structural features(Volz, K. (1993) Biochemistry 32, 11741-11753). The 3-dimensionalstructures of CheY from Salmonella typhimurium (Stock, A. M., Mottonen,J. M., Stock, J. B. & Schutt, ,C. E. (1989) Nature, 337, 745-749) andEscherichia coli (Volz, K. & Matsumura, P. (1991) J. Biol. Chem. 266,15511-15519) and the N-terminal domain of nitrogen regulatory protein Cfrom S. typhimurium (Volkman, B. F., Nohaile, M. J., Amy, N. K., Kustu,S. & Wemmer, D. E. (1995) Biochemistry, 34 1413-1424), are available, aswell as the secondary structure of SpoOF from Bacillus subtilis (Feher,V. A., Zapf, J. W., Hoch, J. A., Dahlquist, F. W., Whiteley, J. M. &Cavanagh, J. (1995) Protein Science, 4, 1801-1814). These structureshave an (α/β)₅ fold. Several structural residues are conserved betweendifferent response regulator sequences, specifically hydrophobicresidues within the β-sheet hydrophobic core and sites from thea-helices. This family of response regulators includes PhoP protein fromBacillus subtilis (Seki, T., Yoshikawa, H, Takahashi, H. & Saito, H., J.Bacteriol., 169, 2913-2916 (1987)).

Histidine kinases are components of the TCSTS which autophosphorylate ahistidine residue. The phosphate group is then transferred to thecognate response regulator. The Histidine kinases have five shortconserved amino acid sequences (Stock, J. B., Ninfa, A. J. & Stock, A.M. (1989) Microbiol. Rev. 53, 450-490, Swanson, R. V., Alex, L. A. &Simon, M. I. (1994) TIBS 19 485-491). These are the histidine residue,which is phosphorylated, followed after approximately 100 residues by aconserved asparagine residue. After another 15 to 45 residues a DXGXGmotif is found, followed by a FXXF motif after another 10-20 residues.10-20 residues further on another glycine motif, GXG is found. The twoglycine motifs are thought to be involved in nucleotide binding.

Among the processes regulated by TCSTS are production of virulencefactors, motility, antibiotic resistance and cell replication.Inhibitors of TCSTS proteins would prevent the bacterium fromestablishing and maintaining infection of the host by preventing it fromproducing the necessary factors for pathogenesis and thereby haveutility in anti-bacterial therapy.

Clearly, there exists a need for polynucleotides and polypeptides, suchas the response regulator embodiments of the invention, that have apresent benefit of, among other things, being useful to screen compoundsfor antibiotic activity. Such factors are also useful to determine theirrole in pathogenesis of infection, dysfunction and disease. There isalso a need for identification and characterization of such factors andtheir antagonists and agonists to find ways to prevent, ameliorate orcorrect such infection, dysfunction and disease.

Certain of the polypeptides of the invention possess significant aminoacid sequence homology to a known PhoP from B. subtilis protein.

SUMMARY OF THE INVENTION

The present invention relates to response regulator, in particularresponse regulator polypeptides and response regulator polynucleotides,recombinant materials and methods for their production. In anotheraspect, the invention relates to methods for using such polypeptides andpolynucleotides, including the treatment of microbial diseases, amongstothers. In a further aspect, the invention relates to methods foridentifying agonists and antagonists using the materials provided by theinvention, and for treating microbial infections and conditionsassociated with such infections with the identified compounds. In astill further aspect, the invention relates to diagnostic assays fordetecting diseases associated with microbial infections and conditionsassociated with such infections, such as assays for detecting responseregulator expression or activity.

Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following descriptions and from reading the otherparts of the present disclosure.

DESCRIPTION OF THE INVENTION

The invention relates to response regulator polypeptides andpolynucleotides as described in greater detail below. In particular, theinvention relates to polypeptides and polynucleotides of a responseregulator of Streptococcus pneumoniae, which is related by amino acidsequence homology to PhoP from B.subtilis polypeptide. The inventionrelates especially to response regulator having the nucleotide and aminoacid sequences set out in Table 1 as SEQ ID NO: 1 or 3 and SEQ ID NO: 2or 4 respectively.

                                      TABLE 1                                     __________________________________________________________________________    Response regulator Polynucleotide and Polypeptide Sequences                   __________________________________________________________________________    (A) Streptococcus pneumoniae response regulator polynucleotide sequence       [SEQ ID NO: 1].                                                                 5' -ATGGGAAAGACAATTTTACTCGTTGACGACGAGGTAGAAATCACAGATATTCATCAGAGA             TGCTTAATTCAGGCAGGTTATCAGGTATTGGTAGCCCAAGATGGACTGGAAGCGATAGAG                  ATGTTCAAGGAAAAACCGATTGATTTGATTATCACAGATGTCATGATGCCTCGGATGGAT                  GGTTATGATTTAATCAGTGAGGTTCAATACTTATCACCAGGGCAGCCTTTCCTATTTATT                  ACTGCTAAGACCAGTGAACAGGACAAGATTTACGGCCTGAGCTTGGGAGCAGATGAATTT                  ATTGCTAAGCCTTTTAGCCCACGTGAGCTGGTTTTGCGTGTCCACAATATTTTGCGCCGC                  CTTCATCGTGGGGGCGAAACAGAGCTGATTTCCCTTGGCAATCTAAAAATGAATCATAGT                  AGTCATGAAGTTCAAATAGGAGAAAAAATGCTGGATTTAACTGTTAAATCATTTGAATTG                  CTGTGGATTTTAGCTAGCAATCCAGAGCGAGTTTTCTCCAAGACAGACCTCTATGAAAAG                  ATCTGGAAAGAAGACTACGTGGATGACACCAATACCTTGAATGTGCATATCCATGCTCTT                  CGACAGGAGCTGGCAAAATATAGTAGTGACCAAACGCCCACTATTAAGACAGTTTGGGGG                  TTGGGATATAAGATAGAGAAACCGAGAGGACAAACATGAAACTAAAAAGTTATATTTTGG                  TTGGATATATTATTTCAACCCTCTTAACCATTTTGGTTGTTTTTTGGGCTGTTCAAAAAA                  TGCTGATTGCGAAAGGCGAGATTTACTTTTTGCTTGGGATGACCATCGTTGCCAGCCTTG                  TCGGTGCTGGGATTAGTCTCTTTCTCCTATTGCCAGTCTTTACGTCGTTGGGCAAACTCA                  AGGAGCATGCCAAGCGGGTAGCGGCCAAGGATTTTCCTTCAAATTTGGAGGTTCAAGGTC                  CTGTAGAATTTCAGCAATTAGGGCAAACTTTTAATGAGATGTCCCATGATTTGCAGGTAA                  GCTTTGATTCCTTGGAAGAAAGCGAACGAGAAAAGGGCTTGATGATTGCCCAGTTGTCGC                  ATGATATTAAGACCCCTATCACTTCGATCCAAGCGACGGTAGAAGGGATTTTGGATGGGA                  TTATCAAGGAGTCGGAGCAAGCTCATTACTAC-3'                                            - (B) Streptococcus pneumoniae response regulator polypeptide sequence      deduced from a                                                                  polynucleotide sequence in this table [SEQ ID NO: 2].                         NH.sub.2 -MGKTILLVDDEVEITDIHQRCLIQAGYQVLVAQDGLEAIEMFKEKPIDLIITDVMMPRMD       GYDLISEVQYLSPGQPFLFITAKTSEQDKIYGLSLGADEFIAKPFSPRELVLRVHNILRR                  LHRGGETELISLGNLKMNHSSHEVQIGEKMLDLTVKSFELLWILASNPERVFSKTDLYEK                  IWKEDYVDDTNTLNVHIHALRQELAKYSSDQTPTIKTVWGLGYKIEKPRGQT-COOH                      - (C) Streptococcus pneumoniae response regulator ORF sequence [SEQ ID      NO: 3].                                                                         5'-                                                                           GCAGGTTATCAGGTATTGGTAGCCCAAGATGGACTGGAAGCGATAGAGATGTTCAAGGAAAAACCGATTGAT     TTGATTATCACAGATGTCATGATGCCTCGGATGGATGGTTATGATTTAATCAGTGAGGTTCAATACTTATCA      CCAGGGCAGCCTTTCCTATTTATTACTGCTAAGACCAGTGAACAGGACAAGATTTACGGCCTGAGCTTGGGA      GCAGATGAATTTATTGCTAAGCCTTTTAGCCCACGTGAGCTGGTTTTGCGTGTCCACAATATTTTGCGCCGC      CTTCATCGTGGGGGCGAAACAGAGCTGATTTCCCTTGGCAATCTAAAAATGAATCATAGTAGTCATGAAGTT      CAAATAGGAGAAAAAATGCTGGATTTAACTGTTAAATCATTTGAATTGCTGTGGATTTTAGCTAGCAATCCA      GAGCGAGTTTTCTCCAAGACAGACCTCTATGAAAAGATCTGGAAAGAAGACTACGTGGATGACACCAATACC      TTGAATGTGCATATCCATGCTCTTCGACAGGAGCTGGCAAAATATAGTAGTGACCAAACGCCCACTATTAAG      ACAGTTTGGGGGTTGGGATATAAGATAGAGAAACCGAGAGGACAAACATGAAACTAAAAAGTTATATTTTGG      TTGGATATATTATTTCAACCCTCTTAACCATTTTGGTTGTTTTTTGGGCTGTTCAAAAAATGCTGATTGCGA      AAGGCGAGATTTACTTTTTGCTTGGGATGACCATCGTTGCCAGCCTTGTCGGTGCTGGGATTAGTCTCTTTC      TCCTATTGCCAGTCTTTACGTCGTTGGGCAAACTCAAGGAGCATGCCAAGCGGGTAGCGGCCAAGGATTTTC      CTTCAAATTTGGAGGTTCAAGGTCCTGTAGAATTTCAGCAATTAGGGCAAACTTTTAATGAGATGTCCCATG      ATTTGCAGGTAAGCTTTGATTCCTTGGAAGAAAGCGAACGAGAAAAGGGCTTGATGATTGCCCAGTTGTCGC      ATGATATTAAGACCCCTATCACTTCGATCCAAGCGACGGTAGAAGGGATTTTGGATGGGATTATCAAGGAGT      CGGAGCAAGCTCATTACTAC-3'                                                        - (D) Streptococcus pneumoniae response regulator polypeptide sequence      deduced from a                                                                  polynucleotide ORF sequence in this table [SEQ ID NO: 4].                     NH.sub.2 -                                                                    AGYQVLVAQDGLEAIEMFKEKPIDLIITDVMMPRMDGYDLISEVQYLSPGQPFLFITAKTSEQDKIYGLSLG     ADEFIAKPFSPRELVLRVHNILRRLHRGGETELISLGNLKMNHSSHEVQIGEKMLDLTVKSFELLWILASNP      ERVFSKTDLYEKIWKEDYVDDTNTLNVHIHALRQELAKYSSDQTPTIKTVWGLGYKIEKPRGQT-COOH          - (E) Polynucleotide sequence from Streptococcus pneumoniae Histidine       Kinase [SEQ ID NO: 5],                                                          cognate of the Response of the invention.                                     5'-                                                                                 AGATAGAGAAACCGAGAGGACAAACATGAAACTAAAAAGTTATATTTTGGTTGGATATATTATTTC     AACCCTCTTAACCATTTTGGTTGTTTTTTGGGCTGTTCAAAAAATGCTGATTGCGAAAGGCGAGATTTACTT      TTTGCTTGGGATGACCATCGTTGCCAGCCTTGTCGGTGCTGGGATTAGTCTCTTTCTCCTATTGCCAGTCTT      TACGTCGTTGGGCAAACTCAAGGAGCATGCCAAGCGGGTAGCGGCCAAGGATTTTCCTTCAAATTTGGAGGT      TCAAGGTCCTGTAGAATTTCAGCAATTAGGGCAAACTTTTAATGAGATGTCCCATGATTTGCAGGTAAGCTT      TGATTCCTTGGAAGAAAGCGAACGAGAAAAGGGCTTGATGATTGCCCAGTTGTCGCATGATATTAAGACCCC      TATCACTTCGATCCAAGCGACGGTAGAAGGGATTTTGGATGGGATTATCAAGGAGTCGGAGCAAGCTCATTA      TCTAGCAACCATTGGACGCCAGACGGAGAGGCTCAATAAACTGGTTGAGGAGTTGAATTTTTTGACCCTAAA      CACAGCTAGAAATCAGGTGGAAACTACCAGTAAAGACAGTATTTTTCTGGACAAGCTCTTAATTGAGTGCAT      GAGTGAATTTCAGTTTTTGATTGAGCAGGAGAGAAGAGATGTCCACTTGCAGGTAATCCCAGAGTCTGCCCG      GATTGAGGGAGATTATGCTAAGCTTTCTCGTATCTTGGTGAATCTGGTCGATAACGCTTTTAAATATTCTGC      TCCAGGAACCAAGCTGGAAGTGGTGACTAAGCTGGAGAAGGGCCAGCTTTCAATCAGTGTGACCGATGAAGG      GCAGGGCATTGCCCCAGAGGATTTGGAAAATATTTTCAAACGCCTTTATCGTGTCGAAACTTCGCGTAACAT      GAAGACAGGTGGTCATGGATTAGGACTTGCGATTGCGCGTGAATTGGCCCATCAATTGGGTGGGGAAATCAC      AGTCAGCAGCCAGTACGGTCTAGGAAGTACCTTTACCCTCGTTCTCAATCTCTCTGGTAGTGAAAATAAAGC      CTAAAACCCCTTTACAAATCCAG                                                        - (F) Polypeptide sequences from Streptococcus pneumoniae Histidine         Kinase [SEQ ID NO: 6]                                                           deduced from the polynucleotide of SEQ ID NO: 3, cognate of the             Response Regulator of the                                                       invention.                                                                    NH.sub.2 -                                                                    MKLKSYILVGYIISTLLTILVVFWAVQKMLIAKGEIYFLLGMTIVASLVGAGISLFLLLPVFTSLGKLKEHA     KRVAAKDFPSNLEVQGPVEFQQLGQTFNEMSHDLQVSFDSLEESEREKGLMIAQLSHDIKTPITSIQATVEG      ILDGIIKESEQAHYLATIGRQTERLNKLVEELNFLTLNTARNQVETTSKDSIFLDKLLIECMSEFQFLIEQE      RRDVHLQVIPESARIEGDYAKLSRILVNLVDNAFKYSAPGTKLEVVTKLEKGQLSISVTDEGQGIAPEDLEN      IFKRLYRVETSRNMKTGGHGLGLAIARELAHQLGGEITVSSQYGLGSTFTLVLNLSGSENKA-COOH          __________________________________________________________________________

Deposited materials

A deposit containing a Streptococcus pneumoniae 0100993 strain has beendeposited with the National Collections of Industrial and MarineBacteria Ltd. (herein "NCIMB"), 23 St. Machar Drive, Aberdeen AB2 IRY,Scotland on Apr. 11, 1996 and assigned deposit number 40794. The depositwas described as Streptococcus pneumoniae 0100993 on deposit.

On Apr. 17, 1996 a Streptococcus pneumoniae 0100993 DNA library in E.coli was similarly deposited with the NCIMB and assigned deposit number40800. The Streptococcus pneumoniae stain deposit is referred to hereinas "the deposited strain" or as "the DNA of the deposited strain."

The deposited strain contains the full length response regulator gene.The sequence of the polynucleotides contained in the deposited strain,as well as the amino acid sequence of any polypeptide encoded thereby,are controlling in the event of any conflict with any description ofsequences herein.

The deposit of the deposited stain has been made under the terms of theBudapest Treaty on the International Recognition of the Deposit ofMicro-organisms for Purposes of Patent Procedure. The strain will beirrevocably and without restriction or condition released to the publicupon the issuance of a patent. The deposited strain is provided merelyas convenience to those of skill in the art and is not an admission thata deposit is required for enablement, such as that required under 35U.S.C. § 112.

A license may be required to make, use or sell the deposited strain, andcompounds derived therefrom, and no such license is hereby granted.

In one aspect of the invention there is provided an isolated nucleicacid molecule encoding a mature polypeptide expressible by theStreptococcus pneumoniae 0100993 strain, which polypeptide is containedin the deposited strain. Further provided by the invention are responseregulator polynucleotide sequences in the deposited strain, such as DNAand RNA, and amino acid sequences encoded thereby. Also provided by theinvention are response regulator polypeptide and polynucleotidesequences isolated from the deposited strain.

Polypeptides

The Response regulator polypeptide of the invention is substantiallyphylogenetically related to other proteins of the response regulatorfamily.

In one aspect of the invention there are provided polypeptides ofStreptococcus pneumoniae referred to herein as "response regulator" and"response regulator polypeptides" as well as biologically,diagnostically, prophylactically, clinically or therapeutically usefulvariants thereof, and compositions comprising the same.

Among the particularly preferred embodiments of the invention arevariants of response regulator polypeptide encoded by naturallyoccurring alleles of the response regulator gene.

The present invention further provides for an isolated polypeptidewhich:

(a) comprises or consists of an amino acid sequence which has at least70% identity, preferably at least 80% identity, more preferably at least90% identity, yet more preferably at least 95% identity, most preferablyat least 97-99% or exact identity, to that of SEQ ID NO:2 over theentire length of SEQ ID NO:2;

(b) a polypeptide encoded by an isolated polynucleotide comprising orconsisting of a polynucleotide sequence which has at least 70% identity,preferably at least 80% identity, more preferably at least 90% identity,yet more preferably at least 95% identity, even more preferably at least97-99% or exact identity to SEQ ID NO: 1 over the entire length of SEQID NO:1;

(c) a polypeptide encoded by an isolated polynucleotide comprising orconsisting of a polynucleotide sequence encoding a polypeptide which hasat least 70% identity, preferably at least 80% identity, more preferablyat least 90% identity, yet more preferably at least 95% identity, evenmore preferably at least 97-99% or exact identity, to the amino acidsequence of SEQ ID NO:2, over the entire length of SEQ ID NO:2; or

(d) a polypeptide encoded by an isolated polynucleotide comprising orconsisting of a polynucleotide sequence which has at least 70% identity,preferably at least 80% identity, more preferably at least 90% identity,yet more preferably at least 95% identity, even more preferably at least97-99% or exact identity, to SEQ ID NO: 1 over the entire length of SEQID NO:3;

(e) a polypeptide encoded by an isolated polynucleotide comprising orconsisting of a polynucleotide sequence which has at least 70% identity,preferably at least 80% identity, more preferably at least 90% identity,yet more preferably at least 95% identity, even more preferably at least97-99% or exact identity to SEQ ID NO:3 over the entire length of SEQ IDNO:3; or

(f) a polypeptide encoded by an isolated polynucleotide comprising orconsisting of a polynucleotide sequence encoding a polypeptide which hasat least 70% identity, preferably at least 80% identity, more preferablyat least 90% identity, yet more preferably at least 95% identity, evenmore preferably at least 97-99% or exact identity, to the amino acidsequence of SEQ ID NO:4, over the entire length of SEQ ID NO:4;

(g) comprises or consists of an amino acid sequence which has at least70% identity, preferably at least 80% identity, more preferably at least90% identity, yet more preferably at least 95% identity, most preferablyat least 97-99% or exact identity, to the amino acid sequence of SEQ IDNO:2 over the entire length of SEQ ID NO:4.

The polypeptides of the invention include a polypeptide of Table 1 [SEQID NO:2 or 4] (in particular the mature polypeptide) as well aspolypeptides and fragments, particularly those which have the biologicalactivity of response regulator, and also those which have at least 70%identity to a polypeptide of Table 1 [SEQ ID NO: 1 or 3]or the relevantportion, preferably at least 80% identity to a polypeptide of Table 1[SEQ ID NO:2 or 4 and more preferably at least 90% identity to apolypeptide of Table 1 [SEQ ID NO:2 or 4] and still more preferably atleast 95% identity to a polypeptide of Table 1 [SEQ ID NO:2 or 4] andalso include portions of such polypeptides with such portion of thepolypeptide generally containing at least 30 amino acids and morepreferably at least 50 amino acids.

The invention also includes a polypeptide consisting of or comprising apolypeptide of the formula:

    X-(R.sub.1).sub.m -(R.sub.2)-(R.sub.3).sub.n -Y

wherein, at the amino terminus, X is hydrogen, a metal or any othermoiety described herein for modified polypeptides, and at the carboxylterminus, Y is hydrogen, a metal or any other moiety described hereinfor modified polypeptides, R₁ and R₃ are any amino acid residue ormodified amino acid residue, m is an integer between 1 and 1000 or zero,n is an integer between 1 and 1000 or zero, and R₂ is an amino acidsequence of the invention, particularly an amino acid sequence selectedfrom Table 1 or modified forms thereof. In the formula above, R₂ isoriented so that its amino terminal amino acid residue is at the left,covalently bound to R₁, and its carboxy terminal amino acid residue isat the right, covalently bound to R₃. Any stretch of amino acid residuesdenoted by either R₁ or R₃, where m and/or n is greater than 1, may beeither a heteropolymer or a homopolymer, preferably a heteropolymer.Other preferred embodiments of the invention are provided where m is aninteger between 1 and 50, 100 or 500, and n is an integer between 1 and50, 100, or 500.

It is most preferred that a polypeptide of the invention is derived fromStreptococcus pneumoniae, however, it may preferably be obtained fromother organisms of the same taxonomic genus. A polypeptide of theinvention may also be obtained, for example, from organisms of the sametaxonomic family or order.

A fragment is a variant polypeptide having an amino acid sequence thatis entirely the same as part but not all of any amino acid sequence ofany polypeptide of the invention. As with response regulatorpolypeptides, fragments may be "free-standing," or comprised within alarger polypeptide of which they form a part or region, most preferablyas a single continuous region in a single larger polypeptide.

Preferred fragments include, for example, truncation polypeptides havinga portion of an amino acid sequence of Table 1 [SEQ ID NO:2 or 4], or ofvariants thereof, such as a continuous series of residues that includesan amino- and/or carboxyl-terminal amino acid sequence. Degradationforms of the polypeptides of the invention produced by or in a hostcell, particularly a Streptococcus pneumoniae, are also preferred.Further preferred are fragments characterized by structural orfunctional attributes such as fragments that comprise alpha-helix andalpha-helix forming regions, beta-sheet and beta-sheet-forming regions,turn and turn-forming regions, coil and coil-forming regions,hydrophilic regions, hydrophobic regions, alpha amphipathic regions,beta amphipathic regions, flexible regions, surface-forming regions,substrate binding region, and high antigenic index regions.

Further preferred fragments include an isolated polypeptide comprisingan amino acid sequence having at least 15, 20, 30, 40, 50 or 100contiguous amino acids from the amino acid sequence of SEQ ID NO: 2, oran isolated polypeptide comprising an amino acid sequence having atleast 15, 20, 30, 40, 50 or 100 contiguous amino acids truncated ordeleted from the amino acid sequence of SEQ ID NO: 2.

Also preferred are biologically active fragments which are thosefragments that mediate activities of response regulator, including thosewith a similar activity or an improved activity, or with a decreasedundesirable activity. Also included are those fragments that areantigenic or immunogenic in an animal, especially in a human.Particularly preferred are fragments comprising receptors or domains ofenzymes that confer a function essential for viability of Streptococcuspneumoniae or the ability to initiate, or maintain cause Disease in anindividual, particularly a human.

Fragments of the polypeptides of the invention may be employed forproducing the corresponding full-length polypeptide by peptidesynthesis; therefore, these variants may be employed as intermediatesfor producing the full-length polypeptides of the invention.

In addition to the standard single and triple letter representations foramino acids, the term "X" or "Xaa" may also be used in describingcertain polypeptides of the invention. "X" and "Xaa" mean that any ofthe twenty naturally occurring amino acids may appear at such adesignated position in the polypeptide sequence.

Polynucleotides

It is an object of the invention to provide polynucleotides that encoderesponse regulator polypeptides, particularly polynucleotides thatencode the polypeptide herein designated response regulator.

In a particularly preferred embodiment of the invention thepolynucleotide comprises a region encoding response regulatorpolypeptides comprising a sequence set out in Table 1 [SEQ ID NO: 1 or3] which includes a fill length gene, or a variant thereof. TheApplicants believe that this fill length gene is essential to the growthand/or survival of an organism which possesses it, such as Streptococcuspneumoniae.

As a further aspect of the invention there are provided isolated nucleicacid molecules encoding and/or expressing response regulatorpolypeptides and polynucleotides, particularly Streptococcus pneumoniaeresponse regulator polypeptides and polynucleotides, including, forexample, unprocessed RNAs, ribozyme RNAs, mRNAs, cDNAs, genomic DNAs, B-and Z-DNAs. Further embodiments of the invention include biologically,diagnostically, prophylactically, clinically or therapeutically usefulpolynucleotides and polypeptides, and variants thereof, and compositionscomprising the same.

Another aspect of the invention relates to isolated polynucleotides,including at least one fill length gene, that encodes a responseregulator polypeptide having a deduced amino acid sequence of Table 1[SEQ ID NO:2 or 4] and polynucleotides closely related thereto andvariants thereof.

In another particularly preferred embodiment of the invention there is aresponse regulator polypeptide from Streptococcus pneumoniae comprisingor consisting of an amino acid sequence of Table 1 [SEQ ID NO:2 or 4],or a variant thereof.

Using the information provided herein, such as a polynucleotide sequenceset out in Table 1 [SEQ ID NO: 1 or 3], a polynucleotide of theinvention encoding response regulator polypeptide may be obtained usingstandard cloning and screening methods, such as those for cloning andsequencing chromosomal DNA fragments from bacteria using Streptococcuspneumoniae 0100993 cells as starting material, followed by obtaining afull length clone. For example, to obtain a polynucleotide sequence ofthe invention, such as a polynucleotide sequence given in Table 1 [SEQID NO: 1 or 3], typically a library of clones of chromosomal DNA ofStreptococcus pneumoniae 0100993 in E. coli or some other suitable hostis probed with a radiolabeled oligonucleotide, preferably a 17-mer orlonger, derived from a partial sequence. Clones carrying DNA identicalto that of the probe can then be distinguished using stringenthybridization conditions. By sequencing the individual clones thusidentified by hybridization with sequencing primers designed from theoriginal polypeptide or polynucleotide sequence it is then possible toextend the polynucleotide sequence in both directions to determine afull length gene sequence. Conveniently, such sequencing is performed,for example, using denatured double stranded DNA prepared from a plasmidclone. Suitable techniques are described by Maniatis, T., Fritsch, E. F.and Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.;Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).(see in particular Screening By Hybridization 1.90 and SequencingDenatured Double-Stranded DNA Templates 13.70). Direct genomic DNAsequencing may also be performed to obtain a full length gene sequence.Illustrative of the invention, each polynucleotide set out in Table 1[SEQ ID NO:1 or 3] was discovered in a DNA library derived fromStreptococcus pneumoniae 0100993.

Moreover, each DNA sequence set out in Table 1 [SEQ ID NO: 1 or 3]contains an open reading frame encoding a protein having about thenumber of amino acid residues set forth in Table 1 [SEQ ID NO:2 or 4]with a deduced molecular weight that can be calculated using amino acidresidue molecular weight values well known to those skilled in the art.The polynucleotide of SEQ ID NO: 1, between nucleotide number I and thestop codon which begins at nucleotide number 697 of SEQ ID NO:1, encodesthe polypeptide of SEQ ID NO:2.

In a further aspect, the present invention provides for an isolatedpolynucleotide comprising or consisting of:

(a) a polynucleotide sequence which has at least 70% identity,preferably at least 80% identity, more preferably at least 90% identity,yet more preferably at least 95% identity, even more preferably at least97-99% or exact identity to SEQ ID NO:1 over the entire length of SEQ IDNO:1;

(b) a polynucleotide sequence encoding a polypeptide which has at least70% identity, preferably at least 80% identity, more preferably at least90% identity, yet more preferably at least 95% identity, even morepreferably at least 97-99% or 100% exact, to the amino acid sequence ofSEQ ID NO:2, over the entire length of SEQ ID NO:2; or

(c) a nucleotide sequence which has at least 70% identity, preferably atleast 80% identity, more preferably at least 90% identity, yet morepreferably at least 95% identity, even more preferably at least 97-99%or 100% identity, to SEQ ID NO: 1 over the entire length of SEQ ID NO:3;

(d) a nucleotide sequence which has at least 70% identity, preferably atleast 80% identity, more preferably at least 90% identity, yet morepreferably at least 95% identity, even more preferably at least 97-99%or exact identity to SEQ ID NO:3 over the entire length of SEQ ID NO:3;or

(e) a polynucleotide sequence encoding a polypeptide which has at least70% identity, preferably at least 80% identity, more preferably at least90% identity, yet more preferably at least 95% identity, even morepreferably at least 97-99% or exact identity, to the amino acid sequenceof SEQ ID NO:4, over the entire length of SEQ ID NO:4.

A polynucleotide encoding a polypeptide of the present invention,including homologs and orthologs from species other than Streptococcuspneumoniae, may be obtained by a process which comprises the steps ofscreening an appropriate library under stringent hybridizationconditions with a labeled or detectable probe consisting of orcomprising the sequence of SEQ ID NO: 1 or 3 or a fragment thereof; andisolating a full-length gene and/or genomic clones containing saidpolynucleotide sequence.

The invention provides a polynucleotide sequence identical over itsentire length to a coding sequence (open reading frame) in Table 1 [SEQID NO:1 or 3]. Also provided by the invention is a coding sequence for amature polypeptide or a fragment thereof, by itself as well as a codingsequence for a mature polypeptide or a fragment in reading frame withanother coding sequence, such as a sequence encoding a leader orsecretory sequence, a pre-, or pro- or prepro-protein sequence. Thepolynucleotide of the invention may also contain at least one non-codingsequence, including for example, but not limited to at least onenon-coding 5' and 3' sequence, such as the transcribed butnon-translated sequences, termination signals (such as rho-dependent andrho-independent termination signals), ribosome binding sites, Kozaksequences, sequences that stabilize mRNA, introns, and polyadenylationsignals. The polynucleotide sequence may also comprise additional codingsequence encoding additional amino acids. For example, a marker sequencethat facilitates purification of the fused polypeptide can be encoded.In certain embodiments of the invention, the marker sequence is ahexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) anddescribed in Gentz et al., Proc. Natl. Acad. Sci., USA 86: 821-824(1989), or an HA peptide tag (Wilson et al., Cell 37: 767 (1984), bothof which may be useful in purifying polypeptide sequence fused to them.Polynucleotides of the invention also include, but are not limited to,polynucleotides comprising a structural gene and its naturallyassociated sequences that control gene expression.

A preferred embodiment of the invention is a polynucleotide ofconsisting of or comprising nucleotide 1 to the nucleotide immediatelyupstream of or including nucleotide 697 set forth in SEQ ID NO:1 ofTable 1, both of which encode the response regulator polypeptide.

The invention also includes a polynucleotide consisting of or comprisinga polynucleotide of the formula:

    X-(R.sub.1).sub.m -(R.sub.2)-(R.sub.3).sub.n -Y

wherein, at the 5' end of the molecule, X is hydrogen, a metal or amodified nucleotide residue, or together with Y defines a covalent bond,and at the 3' end of the molecule, Y is hydrogen, a metal, or a modifiednucleotide residue, or together with X defines the covalent bond, eachoccurrence of R₁ and R₃ is independently any nucleic acid residue ormodified nucleic acid residue, m is an integer between 1 and 3000 orzero, n is an integer between 1 and 3000 or zero, and R₂ is a nucleicacid sequence or modified nucleic acid sequence of the invention,particularly a nucleic acid sequence selected from Table 1 or a modifiednucleic acid sequence thereof. In the polynucleotide formula above, R₂is oriented so that its 5' end nucleic acid residue is at the left,bound to R₁, and its 3' end nucleic acid residue is at the right, boundto R₃. Any stretch of nucleic acid residues denoted by either R₁ and/orR₂, where m and/or n is greater than 1, may be either a heteropolymer ora homopolymer, preferably a heteropolymer. Where, in a preferredembodiment, X and Y together define a covalent bond, the polynucleotideof the above formula is a closed, circular polynucleotide, which can bea double-stranded polynucleotide wherein the formula shows a firststrand to which the second strand is complementary. In another preferredembodiment m and/or n is an integer between 1 and 1000. Other preferredembodiments of the invention are provided where m is an integer between1 and 50, 100 or 500, and n is an integer between 1 and 50, 100, or 500.

It is most preferred that a polynucleotide of the invention is derivedfrom Streptococcus pneumoniae, however, it may preferably be obtainedfrom other organisms of the same taxonomic genus. A polynucleotide ofthe invention may also be obtained, for example, from organisms of thesame taxonomic family or order.

The term "polynucleotide encoding a polypeptide" as used hereinencompasses polynucleotides that include a sequence encoding apolypeptide of the invention, particularly a bacterial polypeptide andmore particularly a polypeptide of the Streptococcus pneumoniae responseregulator having an amino acid sequence set out in Table 1 [SEQ ID NO:2or 4]. The term also encompasses polynucleotides that include a singlecontinuous region or discontinuous regions encoding the polypeptide (forexample, polynucleotides interrupted by integrated phage, an integratedinsertion sequence, an integrated vector sequence, an integratedtransposon sequence, or due to RNA editing or genomic DNAreorganization) together with additional regions, that also may containcoding and/or non-coding sequences.

The invention further relates to variants of the polynucleotidesdescribed herein that encode variants of a polypeptide having a deducedamino acid sequence of Table 1 [SEQ ID NO:2 or 4]. Fragments of apolynucleotides of the invention may be used, for example, to synthesizefull-length polynucleotides of the invention.

Further particularly preferred embodiments are polynucleotides encodingresponse regulator variants, that have the amino acid sequence ofresponse regulator polypeptide of Table 1 [SEQ ID NO:2 or 4] in whichseveral, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residuesare substituted, modified, deleted and/or added, in any combination.Especially preferred among these are silent substitutions, additions anddeletions, that do not alter the properties and activities of responseregulator polypeptide.

Further preferred embodiments of the invention are polynucleotides thatare at least 70% identical over their entire length to a polynucleotideencoding response regulator polypeptide having an amino acid sequenceset out in Table 1 [SEQ ID NO:2 or 4], and polynucleotides that arecomplementary to such polynucleotides. Alternatively, most highlypreferred are polynucleotides that comprise a region that is at least80% identical over its entire length to a polynucleotide encodingresponse regulator polypeptide and polynucleotides complementarythereto. In this regard, polynucleotides at least 90% identical overtheir entire length to the same are particularly preferred, and amongthese particularly preferred polynucleotides, those with at least 95%are especially preferred. Furthermore, those with at least 97% arehighly preferred among those with at least 95%, and among these thosewith at least 98% and at least 99% are particularly highly preferred,with at least 99% being the more preferred.

Preferred embodiments are polynucleotides encoding polypeptides thatretain substantially the same biological function or activity as themature polypeptide encoded by a DNA of Table 1 [SEQ ID NO:1 or 3].

In accordance with certain preferred embodiments of this invention thereare provided polynucleotides that hybridize, particularly understringent conditions, to response regulator polynucleotide sequences,such as those polynucleotides in Table 1.

The invention further relates to polynucleotides that hybridize to thepolynucleotide sequences provided herein. In this regard, the inventionespecially relates to polynucleotides that hybridize under stringentconditions to the polynucleotides described herein. As herein used, theterms "stringent conditions" and "stringent hybridization conditions"mean hybridization occurring only if there is at least 95% andpreferably at least 97% identity between the sequences. A specificexample of stringent hybridization conditions is overnight incubation at42° C. in a solution comprising: 50% formamide, 5× SSC (150 mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/ml of denatured,sheared salmon sperm DNA, followed by washing the hybridization supportin 0.1× SSC at about 65° C. Hybridization and wash conditions are wellknown and exemplified in Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989),particularly Chapter 11 therein. Solution hybridization may also be usedwith the polynucleotide sequences provided by the invention.

The invention also provides a polynucleotide consisting of or comprisinga polynucleotide sequence obtained by screening an appropriate librarycontaining the complete gene for a polynucleotide sequence set forth inSEQ ID NO:1 or 3 under stringent hybridization conditions with a probehaving the sequence of said polynucleotide sequence set forth in SEQ IDNO: 1 or 3 or a fragment thereof, and isolating said polynucleotidesequence. Fragments useful for obtaining such a polynucleotide include,for example, probes and primers fully described elsewhere herein.

As discussed elsewhere herein regarding polynucleotide assays of theinvention, for instance, the polynucleotides of the invention, may beused as a hybridization probe for RNA, cDNA and genomic DNA to isolatefull-length cDNAs and genomic clones encoding response regulator and toisolate cDNA and genomic clones of other genes that have a highidentity, particularly high sequence identity, to the response regulatorgene. Such probes generally will comprise at least 15 nucleotideresidues or base pairs. Preferably, such probes will have at least 30nucleotide residues or base pairs and may have at least 50 nucleotideresidues or base pairs. Particularly preferred probes will have at least20 nucleotide residues or base pairs and will have lee than 30nucleotide residues or base pairs.

A coding region of a response regulator gene may be isolated byscreening using a DNA sequence provided in Table 1 [SEQ ID NO: 1 or 3]to synthesize an oligonucleotide probe. A labeled oligonucleotide havinga sequence complementary to that of a gene of the invention is then usedto screen a library of cDNA, genomic DNA or mRNA to determine whichmembers of the library the probe hybridizes to.

There are several methods available and well known to those skilled inthe art to obtain full-length DNAs, or extend short DNAs, for examplethose based on the method of Rapid Amplification of cDNA ends (RACE)(see, for example, Frohman, et al., PNAS USA 85: 8998-9002, 1988).Recent modifications of the technique, exemplified by the Marathon™technology (Clontech Laboratories Inc.) for example, have significantlysimplified the search for longer cDNAs. In the Marathon™ technology,cDNAs have been prepared from mRNA extracted from a chosen tissue and an`adaptor` sequence ligated onto each end. Nucleic acid amplification(PCR) is then carried out to amplify the "missing" 5' end of the DNAusing a combination of gene specific and adaptor specificoligonucleotide primers. The PCR reaction is then repeated using"nested" primers, that is, primers designed to anneal within theamplified product (typically an adaptor specific primer that annealsfurther 3' in the adaptor sequence and a gene specific primer thatanneals further 5' in the known gene sequence). The products of thisreaction can then be analyzed by DNA sequencing and a full-length DNAconstructed either by joining the product directly to the existing DNAto give a complete sequence, or carrying out a separate full-length PCRusing the new sequence information for the design of the 5' primer.

The polynucleotides and polypeptides of the invention may be employed,for example, as research reagents and materials for discovery oftreatments of and diagnostics for diseases, particularly human diseases,as further discussed herein relating to polynucleotide assays.

The polynucleotides of the invention that are oligonucleotides derivedfrom a sequence of Table 1 [SEQ ID NOS:1 or 2 or 3 or 4] may be used inthe processes herein as described, but preferably for PCR, to determinewhether or not the polynucleotides identified herein in whole or in partare transcribed in bacteria in infected tissue. It is recognized thatsuch sequences will also have utility in diagnosis of the stage ofinfection and type of infection the pathogen has attained.

The invention also provides polynucleotides that encode a polypeptidethat is the mature protein plus additional amino or carboxyl-terminalamino acids, or amino acids interior to the mature polypeptide (when themature form has more than one polypeptide chain, for instance). Suchsequences may play a role in processing of a protein from precursor to amature form, may allow protein transport, may lengthen or shortenprotein half-life or may facilitate manipulation of a protein for assayor production, among other things. As generally is the case in vivo, theadditional amino acids may be processed away from the mature protein bycellular enzymes.

For each and every polynucleotide of the invention there is provided apolynucleotide complementary to it. It is preferred that thesecomplementary polynucleotides are fully complementary to eachpolynucleotide with which they are complementary.

A precursor protein, having a mature form of the polypeptide fused toone or more prosequences may be an inactive form of the polypeptide.When prosequences are removed such inactive precursors generally areactivated. Some or all of the prosequences may be removed beforeactivation. Generally, such precursors are called proproteins.

In addition to the standard A, G, C, T/U representations fornucleotides, the term "N" may also be used in describing certainpolynucleotides of the invention. "N" means that any of the four DNA orRNA nucleotides may appear at such a designated position in the DNA orRNA sequence, except it is preferred that N is not a nucleic acid thatwhen taken in combination with adjacent nucleotide positions, when readin the correct reading frame, would have the effect of generating apremature termination codon in such reading frame.

In sum, a polynucleotide of the invention may encode a mature protein, amature protein plus a leader sequence (which may be referred to as apreprotein), a precursor of a mature protein having one or moreprosequences that are not the leader sequences of a preprotein, or apreproprotein, which is a precursor to a proprotein, having a leadersequence and one or more prosequences, which generally are removedduring processing steps that produce active and mature forms of thepolypeptide.

Vectors, Host Cells, Expression Systems

The invention also relates to vectors that comprise a polynucleotide orpolynucleotides of the invention, host cells that are geneticallyengineered with vectors of the invention and the production ofpolypeptides of the invention by recombinant techniques. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the invention.

Recombinant polypeptides of the present invention may be prepared byprocesses well known in those skilled in the art from geneticallyengineered host cells comprising expression systems. Accordingly, in afurther aspect, the present invention relates to expression systemswhich comprise a polynucleotide or polynucleotides of the presentinvention, to host cells which are genetically engineered with suchexpression systems, and to the production of polypeptides of theinvention by recombinant techniques.

For recombinant production of the polypeptides of the invention, hostcells can be genetically engineered to incorporate expression systems orportions thereof or polynucleotides of the invention. Introduction of apolynucleotide into the host cell can be effected by methods describedin many standard laboratory manuals, such as Davis, et al., BASICMETHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook, et al., MOLECULARCLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989), such as, calcium phosphatetransfection, DEAE-dextran mediated transfection, transvection,microinjection, cationic lipid-mediated transfection, electroporation,transduction, scrape loading, ballistic introduction and infection.

Representative examples of appropriate hosts include bacterial cells,such as cells of streptococci, staphylococci, enterococci E. coli,streptomyces, cyanobacteria, Bacillus subtilis, and Streptococcuspneumoniae; fungal cells, such as cells of a yeast, Kluveromyces,Saccharomyces, a basidiomycete, Candida albicans and Aspergillus; insectcells such as cells of Drosophila S2 and Spodoptera Sf9; animal cellssuch as CHO, COS, HeLa, C127, 3T3, BHK, 293, CV-1 and Bowes melanomacells; and plant cells, such as cells of a gymnosperm or angiosperm.

A great variety of expression systems can be used to produce thepolypeptides of the invention. Such vectors include, among others,chromosomal-, episomal- and virus-derived vectors, for example, vectorsderived from bacterial plasmids, from bacteriophage, from transposons,from yeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses, picornaviruses and retroviruses, and vectors derived fromcombinations thereof, such as those derived from plasmid andbacteriophage genetic elements, such as cosmids and phagemids. Theexpression system constructs may contain control regions that regulateas well as engender expression. Generally, any system or vector suitableto maintain, propagate or express polynucleotides and/or to express apolypeptide in a host may be used for expression in this regard. Theappropriate DNA sequence may be inserted into the expression system byany of a variety of well-known and routine techniques, such as, forexample, those set forth in Sambrook et al., MOLECULAR CLONING, ALABORATORY MANUAL, (supra).

In recombinant expression systems in eukaryotes, for secretion of atranslated protein into the lumen of the endoplasmic reticulum, into theperiplasmic space or into the extracellular environment, appropriatesecretion signals may be incorporated into the expressed polypeptide.These signals may be endogenous to the polypeptide or they may beheterologous signals.

Polypeptides of the invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography, and lectin chromatography. Most preferably, highperformance liquid chromatography is employed for purification. Wellknown techniques for refolding protein may be employed to regenerateactive conformation when the polypeptide is denatured during isolationand or purification.

Diagnostic, Prognostic, Serotyping and Mutation Assays

This invention is also related to the use of response regulatorpolynucleotides and polypeptides of the invention for use as diagnosticreagents. Detection of response regulator polynucleotides and/orpolypeptides in a eukaryote, particularly a mammal, and especially ahuman, will provide a diagnostic method for diagnosis of disease,staging of disease or response of an infectious organism to drugs.

Eukaryotes, particularly mammals, and especially humans, particularlythose infected or suspected to be infected with an organism comprisingthe response regulator gene or protein, may be detected at the nucleicacid or amino acid level by a variety of well known techniques as wellas by methods provided herein.

Polypeptides and polynucleotides for prognosis, diagnosis or otheranalysis may be obtained from a putatively infected and/or infectedindividual's bodily materials. Polynucleotides from any of thesesources, particularly DNA or RNA, may be used directly for detection ormay be amplified enzymatically by using PCR or any other amplificationtechnique prior to analysis. RNA, particularly mRNA, cDNA and genornicDNA may also be used in the same ways. Using amplification,characterization of the species and strain of infectious or residentorganism present in an individual, may be made by an analysis of thegenotype of a selected polynucleotide of the organism. Deletions andinsertions can be detected by a change in size of the amplified productin comparison to a genotype of a reference sequence selected from arelated organism, preferably a different species of the same genus or adifferent strain of the same species. Point mutations can be identifiedby hybridizing amplified DNA to labeled response regulatorpolynucleotide sequences. Perfectly or significantly matched sequencescan be distinguished from imperfectly or more significantly mismatchedduplexes by DNase or RNase digestion, for DNA or RNA respectively, or bydetecting differences in melting temperatures or renaturation kinetics.Polynucleotide sequence differences may also be detected by alterationsin the electrophoretic mobility of polynucleotide fragments in gels ascompared to a reference sequence. This may be carried out with orwithout denaturing agents. Polynucleotide differences may also bedetected by direct DNA or RNA sequencing. See, for example, Myers etal., Science, 230: 1242 (1985). Sequence changes at specific locationsalso may be revealed by nuclease protection assays, such as RNase, V1and S1 protection assay or a chemical cleavage method. See, for example,Cotton et al., Proc. Natl. Acad. Sci., USA, 85: 43974401(1985).

In another embodiment, an array of oligonucleotides probes comprisingresponse regulator nucleotide sequence or fragments thereof can beconstructed to conduct efficient screening of, for example, geneticmutations, serotype, taxonomic classification or identification. Arraytechnology methods are well known and have general applicability and canbe used to address a variety of questions in molecular geneticsincluding gene expression, genetic linkage, and genetic variability(see, for example, Chee et al, Science, 274: 610 (1996)).

Thus in another aspect, the present invention relates to a diagnostickit which comprises:

(a) a polynucleotide of the present invention, preferably the nucleotidesequence of SEQ ID NO: 1 or 3, or a fragment thereof;

(b) a nucleotide sequence complementary to that of (a);

(c) a polypeptide of the present invention, preferably the polypeptideof SEQ ID NO:2 or 4 or a fragment thereof; or

(d) an antibody to a polypeptide of the present invention, preferably tothe polypeptide of SEQ ID NO:2 or 4.

It will be appreciated that in any such kit, (a), (b), (c) or (d) maycomprise a substantial component. Such a kit will be of use indiagnosing a disease or susceptibility to a Disease, among others.

This invention also relates to the use of polynucleotides of the presentinvention as diagnostic reagents. Detection of a mutated form of apolynucleotide of the invention, preferable, SEQ ID NO:1 or 3, which isassociated with a disease or pathogenicity will provide a diagnostictool that can add to, or define, a diagnosis of a disease, a prognosisof a course of disease, a determination of a stage of disease, or asusceptibility to a disease, which results from under-expression,over-expression or altered expression of the polynucleotide. Organisms,particularly infectious organisms, carrying mutations in suchpolynucleotide may be detected at the polynucleotide level by a varietyof techniques, such as those described elsewhere herein.

The nucleotide sequences of the present invention are also valuable fororganism chromosome identification. The sequence is specificallytargeted to, and can hybridize with, a particular location on anorganism's chromosome, particularly to a Streptococcus pneumoniaechromosome. The mapping of relevant sequences to chromosomes accordingto the present invention may be an important step in correlating thosesequences with pathogenic potential and/or an ecological niche of anorganism and/or drug resistance of an organism, as well as theessentiality of the gene to the organism. Once a sequence has beenmapped to a precise chromosomal location, the physical position of thesequence on the chromosome can be correlated with genetic map data. Suchdata may be found on-line in a sequence database. The relationshipbetween genes and diseases that have been mapped to the same chromosomalregion are then identified through known genetic methods, for example,through linkage analysis (coinheritance of physically adjacent genes) ormating studies, such as by conjugation.

The differences in a polynucleotide and/or polypeptide sequence betweenorganisms possessing a first phenotype and organisms possessing adifferent, second different phenotype can also be determined. If amutation is observed in some or all organisms possessing the firstphenotype but not in any organisms possessing the second phenotype, thenthe mutation is likely to be the causative agent of the first phenotype.

Cells from an organism carrying mutations or polymorphisms (allelicvariations) in a polynucleotide and/or polypeptide of the invention mayalso be detected at the polynucleotide or polypeptide level by a varietyof techniques, to allow for serotyping, for example. For example, RT-PCRcan be used to detect mutations in the RNA. It is particularly preferredto use RT-PCR in conjunction with automated detection systems, such as,for example, GeneScan. RNA, cDNA or genomic DNA may also be used for thesame purpose, PCR. As an example, PCR primers complementary to apolynucleotide encoding response regulator polypeptide can be used toidentify and analyze mutations. Examples of representative primers areshown below in Table 2.

                  TABLE 2                                                         ______________________________________                                        Primers for amplification of response regulator                                polynucleotides                                                                  SEQ ID NO     PRIMER SEQUENCE                                             ______________________________________                                        7             5'-ATGGGAAAGACAATTTTACTCGTTG-3'                                   85'-TGTTTGTCCTCTCGGTTTCTCTATC-3'                                            ______________________________________                                    

The invention also includes primers of the formula:

    X-(R.sub.1).sub.m -(R.sub.2)-(R.sub.3).sub.n -Y

wherein, at the 5' end of the molecule, X is hydrogen, a metal or amodified nucleotide residue, and at the 3' end of the molecule, Y ishydrogen, a metal or a modified nucleotide residue, R₁ and R₃ are anynucleic acid residue or modified nucleotide residue, m is an integerbetween 1 and 20 or zero , n is an integer between 1 and 20 or zero, andR₂ is a primer sequence of the invention, particularly a primer sequenceselected from Table 2. In the polynucleotide formula above R₂ isoriented so that its 5' end nucleotide residue is at the left, bound toR₁, and its 3' end nucleotide residue is at the right, bound to R₃. Anystretch of nucleic acid residues denoted by either R group, where mand/or n is greater than 1, may be either a heteropolymer or ahomopolymer, preferably a heteropolymer being complementary to a regionof a polynucleotide of Table 1. In a preferred embodiment m and/or n isan integer between 1 and 10.

The invention further provides these primers with 1, 2, 3 or 4nucleotides removed from the 5' and/or the 3' end. These primers may beused for, among other things, amplifying response regulator DNA and/orRNA isolated from a sample derived from an individual, such as a bodilymaterial. The primers may be used to amplify a polynucleotide isolatedfrom an infected individual, such that the polynucleotide may then besubject to various techniques for elucidation of the polynucleotidesequence. In this way, mutations in the polynucleotide sequence may bedetected and used to diagnose and/or prognose the infection or its stageor course, or to serotype and/or classify the infectious agent.

The invention further provides a process for diagnosing, disease,preferably bacterial infections, more preferably infections caused byStreptococcus pneumoniae, comprising determining from a sample derivedfrom an individual, such as a bodily material, an increased level ofexpression of polynucleotide having a sequence of Table 1 [SEQ ID NO: 1or 3]. Increased or decreased expression of a response regulatorpolynucleotide can be measured using any on of the methods well known inthe art for the quantitation of polynucleotides, such as, for example,amplification, PCR, RT-PCR, RNase protection, Northern blotting,spectrometry and other hybridization methods.

In addition, a diagnostic assay in accordance with the invention fordetecting over-expression of response regulator polypeptide compared tonormal control tissue samples may be used to detect the presence of aninfection, for example. Assay techniques that can be used to determinelevels of a response regulator polypeptide, in a sample derived from ahost, such as a bodily material, are well-known to those of skill in theart. Such assay methods include radioimmunoassays, competitive-bindingassays, Western Blot analysis, antibody sandwich assays, antibodydetection and ELISA assays.

Differential Expression

The polynucleotides and polynucleotides of the invention may be used asreagents for differental screening methods. There are many differentialscreening and differential display methods known in the art in which thepolynucleotides and polypeptides of the invention may be used. Forexample, the differential display technique is described by Chuang etal., J. Bacteriol. 175:2026-2036 (1993). This method identifies thosegenes which are expressed in an organism by identifying mRNA presentusing randomly-primed RT-PCR. By comparing pre-infection and postinfection profiles, genes up and down regulated during infection can beidentified and the RT-PCR product sequenced and matched to ORF"unknowns."

In Vivo Expression Technology (IVET) is described by Camilli et al.,Proc. Nat'l. Acad. Sci. USA. 91:2634-2638 (1994). IVET identifies genesup-regulated during infection when compared to laboratory cultivation,implying an important role in infection. ORFs identified by thistechnique are implied to have a significant role in infectionestablishment and/or maintenance. In this technique random chromosomalfragments of target organism are cloned upstream of a promoter-lessrecombinase gene in a plasmid vector. This construct is introduced intothe target organism which carries an antibiotic resistance gene flankedby resolvase sites. Growth in the presence of the antibiotic removesfrom the population those fragments cloned into the plasmid vectorcapable of supporting transcription of the recombinase gene andtherefore have caused loss of antibiotic resistance. The resistant poolis introduced into a host and at various times after infection bacteriamay be recovered and assessed for the presence of antibiotic resistance.The chromosomal fragment carried by each antibiotic sensitive bacteriumshould carry a promoter or portion of a gene normally upregulated duringinfection. Sequencing upstream of the recombinase gene allowsidentification of the up regulated gene.

RT-PCR may also be used to analyze gene expression patterns. For RT PCRusing the polynucleotides of the invention, messenger RNA is isolatedfrom bacterial infected tissue, e.g., 48 hour murine lung infections,and the amount of each mRNA species assessed by reverse transcription ofthe RNA sample primed with random hexanucleotides followed by PCR withgene specific primer pairs. The determination of the presence and amountof a particular mRNA species by quantification of the resultant PCRproduct provides information on the bacterial genes which aretranscribed in the infected tissue. Analysis of gene transcription canbe carried out at different times of infection to gain a detailedknowledge of gene regulation in bacterial pathogenesis allowing for aclearer understanding of which gene products represent targets forscreens for antibacterials. Because of the gene specific nature of thePCR primers employed it should be understood that the bacterial mRNApreparation need not be free of mammalian RNA. This allows theinvestigator to carry out a simple and quick RNA preparation frominfected tissue to obtain bacterial mRNA species which are very shortlived in the bacterium (in the order of 2 minute halflives). Optimallythe bacterial mRNA is prepared from infected murine lung tissue bymechanical disruption in the presence of TRIzole (GIBCO-BRL) for veryshort periods of time, subsequent processing according to themanufacturers of TRIzole reagent and DNAase treatment to removecontaminating DNA. Preferably the process is optimized by finding thoseconditions which give a maximum amount of Streptococcus pneumoniae 16Sribosomal RNA as detected by probing Northerns with a suitably labeledsequence specific oligonucleotide probe. Typically a 5' dye labeledprimer is used in each PCR primer pair in a PCR reaction which isterminated optimally between 8 and 25 cycles. The PCR products areseparated on 6% polyacrylamide gels with detection and quantificationusing GeneScanner (manufactured by ABI).

Gridding and Polynucleotide Subtraction

Methods have been described for obtaining information about geneexpression and identity using so called "high density DNA arrays" orgrids. See, e.g., M. Chee et al., Science, 274:610-614 (1996) and otherreferences cited therein. Such gridding assays have been employed toidentify certain novel gene sequences, referred to as Expressed SequenceTags (EST) (Adams et a., Science, 252:1651-1656 (1991)). A variety oftechniques have also been described for identifying particular genesequences on the basis of their gene products. For example, seeInternational Patent Application No. WO91/07087, published May 30, 1991.In addition, methods have been described for the amplification ofdesired sequences. For example, see International Patent Application No.WO91/17271, published Nov. 14, 1991.

The polynucleotides of the invention may be used as components ofpolynucleotide arrays, preferably high density arrays or grids. Thesehigh density arrays are particularly useful for diagnostic andprognostic purposes. For example, a set of spots each comprising adifferent gene, and further comprising a polynucleotide orpolynucleotides of the invention, may be used for probing, such as usinghybridization or nucleic acid amplification, using a probes obtained orderived from a bodily sample, to determine the presence of a particularpolynucleotide sequence or related sequence in an individual. Such apresence may indicate the presence of a pathogen, particularlyStreptococcus pneumoniae, and may be useful in diagnosing and/orprognosing disease or a course of disease. A grid comprising a number ofvariants of the polynucleotide sequence of SEQ ID NO:1 or 3 arepreferred. Also preferred is a comprising a number of variants of apolynucleotide sequence encoding the polypeptide sequence of SEQ ID NO:2or 4.

Antibodies

The polypeptides and polynucleotides of the invention or variantsthereof, or cells expressing the same can be used as immunogens toproduce antibodies immunospecific for such polypeptides orpolynucleotides respectively.

In certain preferred embodiments of the invention there are providedantibodies against response regulator polypeptides or polynucleotides.

Antibodies generated against the polypeptides or polynucleotides of theinvention can be obtained by administering the polypeptides and/orpolynucleotides of the invention, or epitope-bearing fragments of eitheror both, analogues of either or both, or cells expressing either orboth, to an animal, preferably a nonhuman, using routine protocols. Forpreparation of monoclonal antibodies, any technique known in the artthat provides antibodies produced by continuous cell line cultures canbe used. Examples include various techniques, such as those in Kohler,G. and Milstein, C., Nature 256: 495-497 (1975); Kozbor et al.,Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).

Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce single chain antibodies topolypeptides or polynucleotides of this invention. Also, transgenicnice, or other organisms such as other mammals, may be used to expresshumanized antibodies immunospecific to the polypeptides orpolynucleotides of the invention.

Alternatively, phage display technology may be utilized to selectantibody genes with binding activities towards a polypeptide of theinvention either from repertoires of PCR amplified v-genes oflymphocytes from humans screened for possessing anti-response regulatoror from naive libraries (McCafferty, et al., (1990), Nature 348,552-554; Marks, et al., (1992) Biotechnology 10, 779-783). The affinityof these antibodies can also be improved by, for example, chainshuffling (Clackson et al., (1991) Nature 352: 628).

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptides or polynucleotides of the inventionto purify the polypeptides or polynucleotides by, for example, affinitychromatography.

Thus, among others, antibodies against response regulator-polypeptide orresponse regulator-polynucleotide may be employed to treat infections,particularly bacterial infections.

Polypeptide variants include antigenically, epitopically orimmunologically equivalent variants form a particular aspect of thisinvention.

A polypeptide or polynucleotide of the invention, such as anantigenically or immunologically equivalent derivative or a fusionprotein of the polypeptide is used as an antigen to immunize a mouse orother animal such as a rat or chicken. The fusion protein may providestability to the polypeptide. The antigen may be associated, for exampleby conjugation, with an immunogenic carrier protein for example bovineserum albumin, keyhole limpet haemocyanin or tetanus toxoid.Alternatively, a multiple antigenic polypeptide comprising multiplecopies of the polypeptide, or an antigenically or immunologicallyequivalent polypeptide thereof may be sufficiently antigenic to improveimmunogenicity so as to obviate the use of a carrier.

Preferably, the antibody or variant thereof is modified to make it lessimmunogenic in the individual. For example, if the individual is humanthe antibody may most preferably be "humanized," where thecomplimentarily determining region or regions of the hybridoma-derivedantibody has been transplanted into a human monoclonal antibody, forexample as described in Jones et al. (1986), Nature 321, 522-525 orTempest et al., (1991) Biotechnology 9, 266-273.

In accordance with an aspect of the invention, there is provided the useof a polynucleotide of the invention for therapeutic or prophylacticpurposes, in particular genetic immunization. Among the particularlypreferred embodiments of the invention are naturally occurring allelicvariants of response regulator polynucleotides and polypeptides encodedthereby.

The use of a polynucleotide of the invention in genetic immunizationwill preferably employ a suitable delivery method such as directinjection of plasmid DNA into muscles (Wolff et al., Hum Mol Genet(1992) 1: 363, Manthorpe et al., Hum. Gene Ther. (1983) 4: 419),delivery of DNA complexed with specific protein carriers (Wu et al., JBiol Chem. (1989) 264: 16985), coprecipitation of DNA with calciumphosphate (Benvenisty & Reshef, PNAS USA, (1986) 83: 9551),encapsulation of DNA in various forms of liposomes (Kaneda et al.,Science (1989) 243: 375), particle bombardment (Tang et al., Nature(1992) 356:152, Eisenbraun et al., DNA Cell Biol (1993) 12: 791) and invivo infection using cloned retroviral vectors (Seeger et al., PNAS USA(1984) 81: 5849).

Antagonists and Agonists--Assays and Molecules

Polypeptides and polynucleotides of the invention may also be used toassess the binding of small molecule substrates and ligands in, forexample, cells, cell-free preparations, chemical libraries, and naturalproduct mixtures. These substrates and ligands may be natural substratesand ligands or may be structural or functional mimetics. See, e.g.,Coligan et al., Current Protocols in Immunology 1(2): Chapter 5 (1991).

Polypeptides and polynucleotides of the present invention areresponsible for many biological functions, including many diseasestates, in particular the Diseases hereinbefore mentioned. It istherefore desirable to devise screening methods to identify compoundswhich stimulate or which inhibit the function of the polypeptide orpolynucleotide. Accordingly, in a further aspect, the present inventionprovides for a method of screening compounds to identify those whichstimulate or which inhibit the function of a polypeptide orpolynucleotide of the invention, as well as related polypeptides andpolynucleotides. In general, agonists or antagonists may be employed fortherapeutic and prophylactic purposes for such Diseases as hereinbeforementioned. Compounds may be identified from a variety of sources, forexample, cells, cell-free preparations, chemical libraries, and naturalproduct mixtures. Such agonists, antagonists or inhibitors so-identifiedmay be natural or modified substrates, ligands, receptors, enzymes,etc., as the case may be, of response regulator polypeptides andpolynucleotides; or may be structural or functional mimetics thereof(see Coligan et al., Current Protocols in Immunology 1(2):Chapter 5(1991)).

The screening methods may simply measure the binding of a candidatecompound to the polypeptide or polynucleotide, or to cells or membranesbearing the polypeptide or polynucleotide, or a fusion protein of thepolypeptide by means of a label directly or indirectly associated withthe candidate compound. Alternatively, the screening method may involvecompetition with a labeled competitor. Further, these screening methodsmay test whether the candidate compound results in a signal generated byactivation or inhibition of the polypeptide or polynucleotide, usingdetection systems appropriate to the cells comprising the polypeptide orpolynucleotide. Inhibitors of activation are generally assayed in thepresence of a known agonist and the effect on activation by the agonistby the presence of the candidate compound is observed. Constitutivelyactive polypeptide and/or constitutively expressed polypeptides andpolynucleotides may be employed in screening methods for inverseagonists or inhibitors, in the absence of an agonist or inhibitor, bytesting whether the candidate compound results in inhibition ofactivation of the polypeptide or polynucleotide, as the case may be.Further, the screening methods may simply comprise the steps of mixing acandidate compound with a solution containing a polypeptide orpolynucleotide of the present invention, to form a mixture, measuringresponse regulator polypeptide and/or polynucleotide activity in themixture, and comparing the response regulator polypeptide and/orpolynucleotide activity of the mixture to a standard. Fusion proteins,such as those made from Fc portion and response regulator polypeptide,as hereinbefore described, can also be used for high-throughputscreening assays to identify antagonists of the polypeptide of thepresent invention, as well as of phylogenetically and and/orfunctionally related polypeptides (see D. Bennett et al., J MolRecognition, 8:52-58 (1995); and K. Johanson et al., J Biol Chem,270(16):9459-9471 (1995)).

The polynucleotides, polypeptides and antibodies that bind to and/orinteract with a polypeptide of the present invention may also be used toconfigure screening methods for detecting the effect of added compoundson the production of mRNA and/or polypeptide in cells. For example, anELISA assay may be constructed for measuring secreted or cell associatedlevels of polypeptide using monoclonal and polyclonal antibodies bystandard methods known in the art. This can be used to discover agentswhich may inhibit or enhance the production of polypeptide (also calledantagonist or agonist, respectively) from suitably manipulated cells ortissues.

The invention also provides a method of screening compounds to identifythose which enhance (agonist) or block (antagonist) the action ofresponse regulator polypeptides or polynucleotides, particularly thosecompounds that are bacteristatic and/or bactericidal. The method ofscreening may involve high-throughput techniques. For example, to screenfor agonists or antagonists, a synthetic reaction nix, a cellularcompartment, such as a membrane, cell envelope or cell wall, or apreparation of any thereof, comprising response regulator polypeptideand a labeled substrate or ligand of such polypeptide is incubated inthe absence or the presence of a candidate molecule that may be aresponse regulator agonist or antagonist. The ability of the candidatemolecule to agonize or antagonize the response regulator polypeptide isreflected in decreased binding of the labeled ligand or decreasedproduction of product from such substrate. Molecules that bindgratuitously, i.e., without inducing the effects of response regulatorpolypeptide are most likely to be good antagonists. Molecules that bindwell and, as the case may be, increase the rate of product productionfrom substrate, increase signal transduction, or increase chemicalchannel activity are agonists. Detection of the rate or level of, as thecase may be, production of product from substrate, signal transduction,or chemical channel activity may be enhanced by using a reporter system.Reporter systems that may be useful in this regard include but are notlimited to colorimetric, labeled substrate converted into product, areporter gene that is responsive to changes in response regulatorpolynucleotide or polypeptide activity, and binding assays known in theart.

Polypeptides of the invention may be used to identify membrane bound orsoluble receptors, if any, for such polypeptide, through standardreceptor binding techniques known in the art. These techniques include,but are not limited to, ligand binding and crosslinking assays in whichthe polypeptide is labeled with a radioactive isotope (for instance, ¹²⁵I), chemically modified (for instance, biotinylated), or fused to apeptide sequence suitable for detection or purification, and incubatedwith a source of the putative receptor (e.g., cells, cell membranes,cell supernatants, tissue extracts, bodily materials). Other methodsinclude biophysical techniques such as surface plasmon resonance andspectroscopy. These screening methods may also be used to identifyagonists and antagonists of the polypeptide which compete with thebinding of the polypeptide to its receptor(s), if any. Standard methodsfor conducting such assays are well understood in the art.

The fluorescence polarization value for a fluorescently-tagged moleculedepends on the rotational correlation time or tumbling rate. Proteincomplexes, such as formed by response regulator polypeptide associatingwith another response regulator polypeptide or other polypeptide,labeled to comprise a fluorescently-labeled molecule will have higherpolarization values than a fluorescently labeled monomeric protein. Itis preferred that this method be used to characterize small moleculesthat disrupt polypeptide complexes.

Fluorescence energy transfer may also be used characterize smallmolecules that interfere with the formation of response regulatorpolypeptide dimers, trimers, tetramers or higher order structures, orstructures formed by response regulator polypeptide bound to anotherpolypeptide. Response regulator polypeptide can be labeled with both adonor and acceptor fluorophore. Upon mixing of the two labeled speciesand excitation of the donor fluorophore, fluorescence energy transfercan be detected by observing fluorescence of the acceptor. Compoundsthat block dimerization will inhibit fluorescence energy transfer.

Surface plasmon resonance can be used to monitor the effect of smallmolecules on response regulator polypeptide self-association as well asan association of response regulator polypeptide and another polypeptideor small molecule. response regulator polypeptide can be coupled to asensor chip at low site density such that covalently bound moleculeswill be monomeric. Solution protein can then passed over the responseregulator polypeptide-coated surface and specific binding can bedetected in real-time by monitoring the change in resonance angle causedby a change in local refractive index. This technique can be used tocharacterize the effect of small molecules on kinetic rates andequilibrium binding constants for response regulator polypeptideself-association as well as an association of response regulatorpolypeptide and another polypeptide or small molecule.

A scintillation proximity assay may be used to characterize theinteraction between an association of response regulator polypeptidewith another response regulator polypeptide or a different polypeptideresponse regulator polypeptide can be coupled to a scintillation-filledbead. Addition of radio-labeled response regulator polypeptide resultsin binding where the radioactive source molecule is in close proximityto the scintillation fluid. Thus, signal is emitted upon responseregulator polypeptide binding and compounds that prevent responseregulator polypeptide self-association or an association of responseregulator polypeptide and another polypeptide or small molecule willdiminish signal.

ICS biosensors have been described by AMBRI (Australian MembraneBiotechnology Research Institute). They couple the self-association ofmacromolecules to the closing of gramacidin-facilitated ion channels insuspended membrane bilayers and hence to a measurable change in theadmittance (similar to impedance) of the biosensor. This approach islinear over six decades of admittance change and is ideally suited forlarge scale, high through-put screening of small molecule combinatoriallibraries.

In other embodiments of the invention there are provided methods foridentifying compounds which bind to or otherwise interact with andinhibit or activate an activity or expression of a polypeptide and/orpolynucleotide of the invention comprising: contacting a polypeptideand/or polynucleotide of the invention with a compound to be screenedunder conditions to permit binding to or other interaction between thecompound and the polypeptide and/or polynucleotide to assess the bindingto or other interaction with the compound, such binding or interactionpreferably being associated with a second component capable of providinga detectable signal in response to the binding or interaction of thepolypeptide and/or polynucleotide with the compound; and determiningwhether the compound binds to or otherwise interacts with and activatesor inhibits an activity or expression of the polypeptide and/orpolynucleotide by detecting the presence or absence of a signalgenerated from the binding or interaction of the compound with thepolypeptide and/or polynucleotide.

Another example of an assay for response regulator agonists is acompetitive assay that combines response regulator and a potentialagonist with response regulator-binding molecules, recombinant responseregulator binding molecules, natural substrates or ligands, or substrateor ligand mimetics, under appropriate conditions for a competitiveinhibition assay. response regulator can be labeled, such as byradioactivity or a colorimetric compound, such that the number ofresponse regulator molecules bound to a binding molecule or converted toproduct can be determined accurately to assess the effectiveness of thepotential antagonist.

Potential antagonists include, among others, small organic molecules,peptides, polypeptides and antibodies that bind to a polynucleotideand/or polypeptide of the invention and thereby inhibit or extinguishits activity or expression. Potential antagonists also may be smallorganic molecules, a peptide, a polypeptide such as a closely relatedprotein or antibody that binds the same sites on a binding molecule,such as a binding molecule, without inducing response regulator-inducedactivities, thereby preventing the action or expression of responseregulator polypeptides and/or polynucleotides by excluding responseregulator polypeptides and/or polynucleotides from binding.

Potential antagonists include a small molecule that binds to andoccupies the binding site of the polypeptide thereby preventing bindingto cellular binding molecules, such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall organic molecules, peptides or peptide-like molecules. Otherpotential antagonists include antisense molecules (see Okano, J.Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORSOF GENE EXPRESSION, CRC Press, Boca Raton, Fla. (1988), for adescription of these molecules). Preferred potential antagonists includecompounds related to and variants of response regulator.

Other examples of potential polypeptide antagonists include antibodiesor, in some cases, oligonucleotides or proteins which are closelyrelated to the ligands, substrates, receptors, enzymes, etc., as thecase may be, of the polypeptide, e.g., a fragment of the ligands,substrates, receptors, enzymes, etc.; or small molecules which bind tothe polypeptide of the present invention but do not elicit a response,so that the activity of the polypeptide is prevented.

Certain of the polypeptides of the invention are biomimetics, functionalmimetics of the natural response regulator polypeptide. These functionalmimetics may be used for, among other things, antagonizing the activityof response regulator polypeptide or as a antigen or immunogen in amanner described elsewhere herein. Functional mimetics of thepolypeptides of the invention include but are not limited to truncatedpolypeptides. For example, preferred functional mimetics include, apolypeptide comprising the polypeptide sequence set forth in SEQ ID NO:2lacking 20, 30, 40, 50, 60, 70 or 80 ammo- or carboxy-terminal aminoacid residues, including fusion proteins comprising one or more of thesetruncated sequences. Polynucleotides encoding each of these functionalmimetics may be used as expression cassettes to express each mimeticpolypeptide. It is preferred that these cassettes comprise 5' and 3'restriction sites to allow for a convenient means to ligate thecassettes together when desired. It is further preferred that thesecassettes comprise gene expression signals known in the art or describedelsewhere herein.

Thus, in another aspect, the present invention relates to a screeningkit for identifying agonists, antagonists, ligands, receptors,substrates, enzymes, etc. for a polypeptide and/or polynucleotide of thepresent invention; or compounds which decrease or enhance the productionof such polypeptides and/or polynucleotides , which comprises:

(a) a polypeptide and/or a polynucleotide of the present invention;

(b) a recombinant cell expressing a polypeptide and/or polynucleotide ofthe present invention;

(c) a cell membrane expressing a polypeptide and/or polynucleotide ofthe present invention; or

(d) antibody to a polypeptide and/or polynucleotide of the presentinvention;

which polypeptide is preferably that of SEQ ID NO:2, and whichpolynucleotide is preferably that of SEQ ID NO:1.

It will be appreciated that in any such kit, (a), (b), (c) or (d) maycomprise a substantial component.

It will be readily appreciated by the skilled artisan that a polypeptideand/or polynucleotide of the present invention may also be used in amethod for the structure-based design of an agonist, antagonist orinhibitor of the polypeptide and/or polynucleotide, by:

(a) determining in the first instance the three-dimensional structure ofthe polypeptide and/or polynucleotide, or complexes thereof;

(b) deducing the three-dimensional structure for the likely reactivesite(s), binding site(s) or motif(s) of an agonist, antagonist orinhibitor;

(c) synthesizing candidate compounds that are predicted to bind to orreact with the deduced binding site(s), reactive site(s), and/ormotif(s); and

(d) testing whether the candidate compounds are indeed agonists,antagonists or inhibitors.

It will be further appreciated that this will normally be an iterativeprocess, and this iterative process may be performed using automated andcomputer-controlled steps.

In a further aspect, the present invention provides methods of treatingabnormal conditions such as, for instance, a Disease, related to eitheran excess of, an under-expression of, an elevated activity of, or adecreased activity of response regulator polypeptide and/orpolynucleotide.

If the expression and/or activity of the polypeptide and/orpolynucleotide is in excess, several approaches are available. Oneapproach comprises administering to an individual in need thereof aninhibitor compound (antagonist) as herein described, optionally incombination with a pharmaceutically acceptable carrier, in an amounteffective to inhibit the function and/or expression of the polypeptideand/or polynucleotide, such as, for example, by blocking the binding ofligands, substrates, receptors, enzymes, etc., or by inhibiting a secondsignal, and thereby alleviating the abnormal condition. In anotherapproach, soluble forms of the polypeptides still capable of binding theligand, substrate, enzymes, receptors, etc. in competition withendogenous polypeptide and/or polynucleotide may be administered.Typical examples of such competitors include fragments of the responseregulator polypeptide and/or polypeptide.

In a further aspect, the present invention relates to geneticallyengineered soluble fusion proteins comprising a polypeptide of thepresent invention, or a fragment thereof, and various portions of theconstant regions of heavy or light chains of immunoglobulins of varioussubclasses (IgG, IgM, IgA, IgE). Preferred as an immunoglobulin is theconstant part of the heavy chain of human IgG, particularly IgG1, wherefusion takes place at the hinge region. In a particular embodiment, theFc part can be removed simply by incorporation of a cleavage sequencewhich can be cleaved with blood clotting factor Xa. Furthermore, thisinvention relates to processes for the preparation of these fusionproteins by genetic engineering, and to the use thereof for drugscreening, diagnosis and therapy. A further aspect of the invention alsorelates to polynucleotides encoding such fusion proteins. Examples offusion protein technology can be found in International PatentApplication Nos. WO94/29458 and WO94/22914.

In still another approach, expression of the gene encoding endogenousresponse regulator polypeptide can be inhibited using expressionblocking techniques. This blocking may be targeted against any step ingene expression, but is preferably targeted against transcription and/ortranslation. An examples of a known technique of this sort involve theuse of antisense sequences, either internally generated or separatelyadministered (see, for example, O° Connor, J Neurochem (1991) 56:560 inOligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988)). Alternatively, oligonucleotides whichform triple helices with the gene can be supplied (see, for example, Leeet al., Nucleic Acids Res (1979) 6:3073; Cooney et al., Science (1988)241:456; Dervan et al., Science (1991) 251:1360). These oligomers can beadministered per se or the relevant oligomers can be expressed in vivo.

Each of the polynucleotide sequences provided herein may be used in thediscovery and development of antibacterial compounds. The encodedprotein, upon expression, can be used as a target for the screening ofantibacterial drugs. Additionally, the polynucleotide sequences encodingthe amino terminal regions of the encoded protein or Shine-Delgarno orother translation facilitating sequences of the respective mRNA can beused to construct antisense sequences to control the expression of thecoding sequence of interest.

The invention also provides the use of the polypeptide, polynucleotide,agonist or antagonist of the invention to interfere with the initialphysical interaction between a pathogen or pathogens and a eukaryotic,preferably mammalian, host responsible for sequelae of infection. Inparticular, the molecules of the invention may be used: in theprevention of adhesion of bacteria, in particular gram positive and/orgram negative bacteria, to eukaryotic, preferably mammalian,extracellular matrix proteins on in-dwelling devices or to extracellularmatrix proteins in wounds; to block response regulator protein-mediatedmammalian cell invasion by, for example, initiating phosphorylation ofmammalian tyrosine kinases (Rosenshine et al., Infect. Immun. 60:2211(1992); to block bacterial adhesion between eukaryotic, preferablymammalian, extracellular matrix proteins and bacterial responseregulator proteins that mediate tissue damage and/or; to block thenormal progression of pathogenesis in infections initiated other than bythe implantation of in-dwelling devices or by other surgical techniques.

This invention provides a method of screening drugs to identify thosewhich

i) interfere with the interaction of the response regulator with ahistidine kinase, the method comprising incubating the responseregulator with histidine kinase in the presence of the drug andmeasuring the ability of the drug to block this interaction;

ii) interfere with the ability of the response regulator to catalyse thetransfer of phosphate group from the histidine kinase to itself, themethod comprising incubating the response regulator with drug andmeasuring the ability of the response regulator to catalyse the removalof phosphate from phosphorylated histidine kinase; and/or

iii) interfere with the ability of the molecule to autodephosphorylateitself once the phosphate had been transferred, the method comprisingincubating the phosphorylated response regulator with drug and measuringthe ability of the response regulator to catalyse theautodephosphorylation.

The histidine kinase is preferably the cognate histidine kinase of theresponse regulator, or another histidine kinase which is capable ofacting as a substrate for the response regulator, and may be fromStreptococcus pneumoniae or another microorganism e.g. Bacillus.Polypeptide and polynucleotide sequences of the cognate kinase of theResponse Regulator of the invention are set forth in Table 1 (E and F).This novel histidine kinase shows 32% identity to the PhoR sensorprotein from B. subtilis.

In accordance with yet another aspect of the invention, there areprovided response regulator agonists and antagonists, preferablybacteristatic or bactericidal agonists and antagonists.

The antagonists and agonists of the invention may be employed, forinstance, to prevent, inhibit and/or treat diseases.

Helicobacter pylori (herein "H. pylori") bacteria infect the stomachs ofover one-third of the world's population causing stomach cancer, ulcers,and gastritis (International Agency for Research on Cancer(1994)Schistosomes, Liver Flukes and Helicobacter Pylori (InternationalAgency for Research on Cancer, Lyon, France,http://www.uicc.ch/ecp/ecp2904.htm). Moreover, the International Agencyfor Research on Cancer recently recognized a cause-and-effectrelationship between H. pylori and gastric adenocarcinoma, classifyingthe bacterium as a Group I (definite) carcinogen. Preferredantimicrobial compounds of the invention (agonists and antagonists ofresponse regulator polypeptides and/or polynucleotides) found usingscreens provided by the invention, or known in the art, particularlynarrow-spectrum antibiotics, should be useful in the treatment of H.pylori infection. Such treatment should decrease the advent of H.pylori-induced cancers, such as gastrointestinal carcinoma. Suchtreatment should also prevent, inhibit and/or cure gastric ulcers andgastritis.

Vaccines

There are provided by the invention, products, compositions and methodsfor assessing response regulator expression, treating disease, assayinggenetic variation, and administering a response regulator polypeptideand/or polynucleotide to an organism to raise an immunological responseagainst a bacteria, especially a Streptococcus pneumoniae bacteria.

Another aspect of the invention relates to a method for inducing animmunological response in an individual, particularly a mammal whichcomprises inoculating the individual with response regulatorpolynucleotide and/or polypeptide, or a fragment or variant thereof,adequate to produce antibody and/or T cell immune response to protectsaid individual from infection, particularly bacterial infection andmost particularly Streptococcus pneumoniae infection. Also provided aremethods whereby such immunological response slows bacterial replication.Yet another aspect of the invention relates to a method of inducingimmunological response in an individual which comprises delivering tosuch individual a nucleic acid vector, sequence or ribozyme to directexpression of response regulator polynucleotide and/or polypeptide, or afragment or a variant thereof, for expressing response regulatorpolynucleotide and/or polypeptide, or a fragment or a variant thereof invivo in order to induce an immunological response, such as, to produceantibody and/or T cell immune response, including, for example,cytokine-producing T cells or cytotoxic T cells, to protect saidindividual, preferably a human, from disease, whether that disease isalready established within the individual or not. One example ofadministering the gene is by accelerating it into the desired cells as acoating on particles or otherwise. Such nucleic acid vector may compriseDNA, RNA, a ribozyme, a modified nucleic acid, a DNA/RNA hybrid, aDNA-protein complex or an RNA-protein complex.

A further aspect of the invention relates to an immunologicalcomposition that when introduced into an individual, preferably a human,capable of having induced within it an immunological response, inducesan immunological response in such individual to a response regulatorpolynucleotide and/or polypeptide encoded therefrom, wherein thecomposition comprises a recombinant response regulator polynucleotideand/or polypeptide encoded therefrom and/or comprises DNA and/or RNAwhich encodes and expresses an antigen of said response regulatorpolynucleotide, polypeptide encoded therefrom, or other polypeptide ofthe invention. The immunological response may be used therapeutically orprophylactically and may take the form of antibody immunity and/orcellular immunity, such as cellular immunity arising from CTL or CD4+ Tcells.

A response regulator polypeptide or a fragment thereof may be fused withco-protein or chemical moiety which may or may not by itself produceantibodies, but which is capable of stabilizing the first protein andproducing a fused or modified protein which will have antigenic and/orimmunogenic properties, and preferably protective properties. Thus fusedrecombinant protein, preferably further comprises an antigenicco-protein, such as lipoprotein D from Hemophilus influenzae,Glutathione-S-transferase (GST) or beta-galactosidase, or any otherrelatively large co-protein which solubilizes the protein andfacilitates production and purification thereof. Moreover, theco-protein may act as an adjuvant in the sense of providing ageneralized stimulation of the immune system of the organism receivingthe protein. The co-protein may be attached to either the amino- orcarboxy-terminus of the first protein.

Provided by this invention are compositions, particularly vaccinecompositions, and methods comprising the polypeptides and/orpolynucleotides of the invention and immunostimulatory DNA sequences,such as those described in Sato, Y. et al. Science 273: 352 (1996).

Also, provided by this invention are methods using the describedpolynucleotide or particular fragments thereof, which have been shown toencode non-variable regions of bacterial cell surface proteins, inpolynucleotide constructs used in such genetic immunization experimentsin animal models of infection with Streptococcus pneumoniae. Suchexperiments will be particularly useful for identifying protein epitopesable to provoke a prophylactic or therapeutic immune response. It isbelieved that this approach will allow for the subsequent preparation ofmonoclonal antibodies of particular value, derived from the requisiteorgan of the animal successfully resisting or clearing infection, forthe development of prophylactic agents or therapeutic treatments ofbacterial infection, particularly Streptococcus pneumoniae infection, inmammals, particularly humans.

A polypeptide of the invention may be used as an antigen for vaccinationof a host to produce specific antibodies which protect against invasionof bacteria, for example by blocking adherence of bacteria to damagedtissue. Examples of tissue damage include wounds in skin or connectivetissue caused, for example, by mechanical, chemical, thermal orradiation damage or by implantation of indwelling devices, or wounds inthe mucous membranes, such as the mouth, throat, mammary glands, urethraor vagina.

The invention also includes a vaccine formulation which comprises animmunogenic recombinant polypeptide and/or polynucleotide of theinvention together with a suitable carrier, such as a pharmaceuticallyacceptable carrier. Since the polypeptides and polynucleotides may bebroken down in the stomach, each is preferably administeredparenterally, including, for example, administration that issubcutaneous, intramuscular, intravenous, or intradermal. Formulationssuitable for parenteral administration include aqueous and non-aqueoussterile injection solutions which may contain anti-oxidants, buffers,bacteristatic compounds and solutes which render the formulationisotonic with the bodily fluid, preferably the blood, of the individual;and aqueous and non-aqueous sterile suspensions which may includesuspending agents or thickening agents. The formulations may bepresented in unit-dose or multi-dose containers, for example, sealedampoules and vials and may be stored in a freeze-dried conditionrequiring only the addition of the sterile liquid carrier immediatelyprior to use. The vaccine formulation may also include adjuvant systemsfor enhancing the immunogenicity of the formulation, such as oil-inwater systems and other systems known in the art. The dosage will dependon the specific activity of the vaccine and can be readily determined byroutine experimentation.

While the invention has been described with reference to certainresponse regulator polypeptides and polynucleotides, it is to beunderstood that this covers fragments of the naturally occurringpolypeptides and polynucleotides, and similar polypeptides andpolynucleotides with additions, deletions or substitutions which do notsubstantially affect the immunogenic properties of the recombinantpolypeptides or polynucleotides.

Compositions, kits and administration

In a further aspect of the invention there are provided compositionscomprising a response regulator polynucleotide and/or a responseregulator polypeptide for administration to a cell or to a multicellularorganism.

The invention also relates to compositions comprising a polynucleotideand/or a polypeptides discussed herein or their agonists or antagonists.The polypeptides and polynucleotides of the invention may be employed incombination with a non-sterile or sterile carrier or carriers for usewith cells, tissues or organisms, such as a pharmaceutical carriersuitable for administration to an individual. Such compositionscomprise, for instance, a media additive or a therapeutically effectiveamount of a polypeptide and/or polynucleotide of the invention and apharmaceutically acceptable carrier or excipient. Such carriers mayinclude, but are not limited to, saline, buffered saline, dextrose,water, glycerol, ethanol and combinations thereof. The formulationshould suit the mode of administration. The invention further relates todiagnostic and pharmaceutical packs and kits comprising one or morecontainers filled with one or more of the ingredients of theaforementioned compositions of the invention.

Polypeptides, polynucleotides and other compounds of the invention maybe employed alone or in conjunction with other compounds, such astherapeutic compounds.

The pharmaceutical compositions may be administered in any effective,convenient manner including, for instance, administration by topical,oral, anal, vaginal, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal or intradermal routes among others.

In therapy or as a prophylactic, the active agent may be administered toan individual as an injectable composition, for example as a sterileaqueous dispersion, preferably isotonic.

Alternatively the composition may be formulated for topical applicationfor example in the form of ointments, creams, lotions, eye ointments,eye drops, ear drops, mouthwash, impregnated dressings and sutures andaerosols, and may contain appropriate conventional additives, including,for example, preservatives, solvents to assist drug penetration, andemollients in ointments and creams. Such topical formulations may alsocontain compatible conventional carriers, for example cream or ointmentbases, and ethanol or oleyl alcohol for lotions. Such carriers mayconstitute from about 1% to about 98% by weight of the formulation; moreusually they will constitute up to about 80% by weight of theformulation.

In a further aspect, the present invention provides for pharmaceuticalcompositions comprising a therapeutically effective amount of apolypeptide and/or polynucleotide, such as the soluble form of apolypeptide and/or polynucleotide of the present invention, agonist orantagonist peptide or small a molecule compound, in combination with apharmaceutically acceptable carrier or excipient. Such carriers include,but are not limited to, saline, buffered saline, dextrose, water,glycerol, ethanol, and combinations thereof. The invention furtherrelates to pharmaceutical packs and kits comprising one or morecontainers filled with one or more of the ingredients of theaforementioned compositions of the invention. Polypeptides,polynucleotides and other compounds of the present invention may beemployed alone or in conjunction with other compounds, such astherapeutic compounds.

The composition will be adapted to the route of administration, forinstance by a systemic or an oral route. Preferred forms of systemicadministration include injection, typically by intravenous injection.Other injection routes, such as subcutaneous, intramuscular, orintraperitoneal, can be used. Alternative means for systemicadministration include transmucosal and transdermal administration usingpenetrants such as bile salts or fusidic acids or other detergents. Inaddition, if a polypeptide or other compounds of the present inventioncan be formulated in an enteric or an encapsulated formulation, oraladministration may also be possible. Administration of these compoundsmay also be topical and/or localized, in the form of salves, pastes,gels, and the like.

For administration to mammals, and particularly humans, it is expectedthat the daily dosage level of the active agent will be from 0.01 mg/kgto 10 mg/kg, typically around 1 mg/kg. The physician in any event willdetermine the actual dosage which will be most suitable for anindividual and will vary with the age, weight and response of theparticular individual. The above dosages are exemplary of the averagecase. There can, of course, be individual instances where higher orlower dosage ranges are merited, and such are within the scope of thisinvention.

In-dwelling devices include surgical implants, prosthetic devices andcatheters, i.e., devices that are introduced to the body of anindividual and remain in position for an extended time. Such devicesinclude, for example, artificial joints, heart valves, pacemakers,vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinarycatheters, continuous ambulatory peritoneal dialysis (CAPD) catheters.

The composition of the invention may be administered by injection toachieve a systemic effect against relevant bacteria shortly beforeinsertion of an in-dwelling device. Treatment may be continued aftersurgery during the in-body time of the device. In addition, thecomposition could also be used to broaden perioperative cover for anysurgical technique to prevent bacterial wound infections, especiallyStreptococcus pneumoniae wound infections.

Many orthopedic surgeons consider that humans with prosthetic jointsshould be considered for antibiotic prophylaxis before dental treatmentthat could produce a bacteremia. Late deep infection is a seriouscomplication sometimes leading to loss of the prosthetic joint and isaccompanied by significant morbidity and mortality. It may therefore bepossible to extend the use of the active agent as a replacement forprophylactic antibiotics in this situation.

In addition to the therapy described above, the compositions of thisinvention may be used generally as a wound treatment agent to preventadhesion of bacteria to matrix proteins exposed in wound tissue and forprophylactic use in dental treatment as an alternative to, or inconjunction with, antibiotic prophylaxis.

Alternatively, the composition of the invention may be used to bathe anindwelling device immediately before insertion. The active agent willpreferably be present at a concentration of 1 μg/ml to 10 mg/ml forbathing of wounds or indwelling devices.

A vaccine composition is conveniently in injectable form. Conventionaladjuvants may be employed to enhance the immune response. A suitableunit dose for vaccination is 0.5-5 microgram/kg of antigen, and suchdose is preferably administered 1-3 times and with an interval of 1-3weeks. With the indicated dose range, no adverse toxicological effectswill be observed with the compounds of the invention which wouldpreclude their administration to suitable individuals.

Sequence Databases, Sequences in a Tangible Medium, and Algorithms

Polynucleotide and polypeptide sequences form a valuable informationresource with which to determine their 2- and 3-dimensional structuresas well as to identify further sequences of similar homology. Theseapproaches are most easily facilitated by storing the sequence in acomputer readable medium and then using the stored data in a knownmacromolecular structure program or to search a sequence database usingwell known searching tools, such as GCC.

The polynucleotide and polypeptide sequences of the invention areparticularly useful as components in databases useful for searchanalyses as well as in sequence analysis algorithms. As used in thissection entitled "Sequence Databases, Sequences in a Tangible Medium,and Algorithms," and in claims related to this section, the terms"polynucleotide of the invention" and "polynucleotide sequence of theinvention" mean any detectable chemical or physical characteristic of apolynucleotide of the invention that is or may be reduced to or storedin a tangible medium, preferably a computer readable form. For example,chromatographic scan data or peak data, photographic data or scan datatherefrom, called bases, and mass spectrographic data. As used in thissection entitled Databases and Algorithms and in claims related thereto,the terms "polypeptide of the invention" and "polypeptide sequence ofthe invention" mean any detectable chemical or physical characteristicof a polypeptide of the invention that is or may be reduced to or storedin a tangible medium, preferably a computer readable form. For example,chromatographic scan data or peak data, photographic data or scan datatherefrom, and mass spectrographic data.

The invention provides a computer readable medium having stored thereonpolypeptide sequences of the invention and/or polynucleotide sequencesof the invention. For example, a computer readable medium is providedcomprising and having stored thereon a member selected from the groupconsisting of: a polynucleotide comprising the sequence of apolynucleotide of the invention; a polypeptide comprising the sequenceof a polypeptide sequence of the invention; a set of polynucleotidesequences wherein at least one of the sequences comprises the sequenceof a polynucleotide sequence of the invention; a set of polypeptidesequences wherein at least one of the sequences comprises the sequenceof a polypeptide sequence of the invention; a data set representing apolynucleotide sequence comprising the sequence of polynucleotidesequence of the invention; a data set representing a polynucleotidesequence encoding a polypeptide sequence comprising the sequence of apolypeptide sequence of the invention; a polynucleotide comprising thesequence of a polynucleotide sequence of the invention; a polypeptidecomprising the sequence of a polypeptide sequence of the invention; aset of polynucleotide sequences wherein at least one of the sequencescomprises the sequence of a polynucleotide sequence of the invention; aset of polypeptide sequences wherein at least one of said sequencescomprises the sequence of a polypeptide sequence of the invention; adata set representing a polynucleotide sequence comprising the sequenceof a polynucleotide sequence of the invention; a data set representing apolynucleotide sequence encoding a polypeptide sequence comprising thesequence of a polypeptide sequence of the invention. The computerreadable medium can be any composition of matter used to storeinformation or data, including, for example, commercially availablefloppy disks, tapes, chips, hard drives, compact disks, and video disks.

Also provided by the invention are methods for the analysis of charactersequences or strings, particularly genetic sequences or encoded geneticsequences. Preferred methods of sequence analysis include, for example,methods of sequence homology analysis, such as identity and similarityanalysis, RNA structure analysis, sequence assembly, cladistic analysis,sequence motif analysis, open reading frame determination, nucleic acidbase calling, nucleic acid base trimming, and sequencing chromatogrampeak analysis.

A computer based method is provided for performing homologyidentification. This method comprises the steps of providing apolynucleotide sequence comprising the sequence a polynucleotide of theinvention in a computer readable medium; and comparing saidpolynucleotide sequence to at least one polynucleotide or polypeptidesequence to identify homology.

A computer based method is also provided for performing homologyidentification, said method comprising the steps of: providing apolypeptide sequence comprising the sequence of a polypeptide of theinvention in a computer readable medium; and comparing said polypeptidesequence to at least one polynucleotide or polypeptide sequence toidentify homology.

A computer based method is still further provided for polynucleotideassembly, said method comprising the steps of: providing a firstpolynucleotide sequence comprising the sequence of a polynucleotide ofthe invention in a computer readable medium; and screening for at leastone overlapping region between said first polynucleotide sequence and asecond polynucleotide sequence.

A further embodiment of the invention provides a computer based methodfor performing homology identification, said method comprising the stepsof: providing a polynucleotide sequence comprising the sequence of apolynucleotide of the invention in a computer readable medium; andcomparing said polynucleotide sequence to at least one polynucleotide orpolypeptide sequence to identify homology.

A further embodiment of the invention provides a computer based methodfor performing homology identification, said method comprising the stepsof: providing a polypeptide sequence comprising the sequence of apolypeptide of the invention in a computer readable medium; andcomparing said polypeptide sequence to at least one polynucleotide orpolypeptide sequence to identify homology.

A further embodiment of the invention provides a computer based methodfor polynucleotide assembly, said method comprising the steps of:providing a first polynucleotide sequence comprising the sequence of apolynucleotide of the invention in a computer readable medium; andscreening for at least one overlapping region between said firstpolynucleotide sequence and a second polynucleotide sequence.

In another preferred embodiment of the invention there is provided acomputer readable medium having stored thereon a member selected fromthe group consisting of: a polynucleotide comprising the sequence of SEQID NO. 1 or 3; a polypeptide comprising the sequence of SEQ ID NO. 2 or4; a set of polynucleotide sequences wherein at least one of saidsequences comprises the sequence of SEQ ID NO. 1 or 3; a set ofpolypeptide sequences wherein at least one of said sequences comprisesthe sequence of SEQ ID NO. 2 or 4; a data set representing apolynucleotide sequence comprising the sequence of SEQ ID NO. 1 or 3; adata set representing a polynucleotide sequence encoding a polypeptidesequence comprising the sequence of SEQ ID NO. 2 or 4; a polynucleotidecomprising the sequence of SEQ ID NO. 1 or 3; a polypeptide comprisingthe sequence of SEQ ID NO. 2 or 4; a set of polynucleotide sequenceswherein at least one of said sequences comprises the sequence of SEQ IDNO. 1 or 3; a set of polypeptide sequences wherein at least one of saidsequences comprises the sequence of SEQ ID NO. 2 or 4; a data setrepresenting a polynucleotide sequence comprising the sequence of SEQ IDNO. 1 or 3; a data set representing a polynucleotide sequence encoding apolypeptide sequence comprising the sequence of SEQ ID NO. 2 or 4. Afurther preferred embodiment of the invention provides a computer basedmethod for performing homology identification, said method comprisingthe steps of providing a polynucleotide sequence comprising the sequenceof SEQ ID NO. 1 or 3 in a computer readable medium; and comparing saidpolynucleotide sequence to at least one polynucleotide or polypeptidesequence to identify homology.

A still further preferred embodiment of the invention provides acomputer based method for performing homology identification, saidmethod comprising the steps of: providing a polypeptide sequencecomprising the sequence of SEQ ID NO. 2 or 4 in a computer readablemedium; and comparing said polypeptide sequence to at least onepolynucleotide or polypeptide sequence to identify homology.

A further embodiment of the invention provides a computer based methodfor polynucleotide assembly, said method comprising the steps of:providing a first polynucleotide sequence comprising the sequence of SEQID NO. 1 or 3 in a computer readable medium; and screening for at leastone overlapping region between said first polynucleotide sequence and asecond polynucleotide sequence.

A further embodiment of the invention provides a computer based methodfor performing homology identification, said method comprising the stepsof: providing a polynucleotide sequence comprising the sequence of SEQID NO. 1 or 3 in a computer readable medium; and comparing saidpolynucleotide sequence to at least one polynucleotide or polypeptidesequence to identify homology.

A further embodiment of the invention provides a computer based methodfor performing homology identification, said method comprising the stepsof: providing a polypeptide sequence comprising the sequence of SEQ IDNO. 2 or 4 in a computer readable medium; and comparing said polypeptidesequence to at least one polynucleotide or polypeptide sequence toidentify homology.

A further embodiment of the invention provides a computer based methodfor polynucleotide assembly, said method comprising the steps of:providing a first polynucleotide sequence comprising the sequence of SEQID NO. 1 or 3 in a computer readable medium; and screening for at leastone overlapping region between said first polynucleotide sequence and asecond polynucleotide sequence.

All publications and references, including but not limited to patentsand patent applications, cited in this specification are hereinincorporated by reference in their entirety as if each individualpublication or reference were specifically and individually indicated tobe incorporated by reference herein as being fully set forth. Any patentapplication to which this application claims priority is alsoincorporated by reference herein in its entirety in the manner describedabove for publications and references.

Glossary

The following definitions are provided to facilitate understanding ofcertain terms used frequently herein.

"Antibody(ies)" as used herein includes polyclonal and monoclonalantibodies, chimeric, single chain, and humanized antibodies, as well asFab fragments, including the products of an Fab or other immunoglobulinexpression library.

"Antigenically equivalent derivative(s)" as used herein encompasses apolypeptide, polynucleotide, or the equivalent of either which will bespecifically recognized by certain antibodies which, when raised to theprotein, polypeptide or polynucleotide according to the invention,interferes with the immediate physical interaction between pathogen andmammalian host.

"Bispecific antibody(ies)" means an antibody comprising at least twoantigen binding domains, each domain directed against a differentepitope.

"Bodily material(s) means any material derived from an individual orfrom an organism infecting, infesting or inhabiting an individual,including but not limited to, cells, tissues and waste, such as, bone,blood, serum, cerebrospinal fluid, semen, saliva, muscle, cartilage,organ tissue, skin, urine, stool or autopsy materials.

"Disease(s)" means any disease caused by or related to infection by abacteria, including, for example, otitis media, conjunctivitis,pneumonia, bacteremia, meningitis, sinusitis, pleural empyema andendocarditis, and most particularly meningitis, such as for exampleinfection of cerebrospinal fluid.

"Fusion protein(s)" refers to a protein encoded by two, often unrelated,fused genes or fragments thereof. In one example, EP-A-0464 disclosesfusion proteins comprising various portions of constant region ofimmunoglobulin molecules together with another human protein or partthereof. In many cases, employing an immunoglobulin Fc region as a partof a fusion protein is advantageous for use in therapy and diagnosisresulting in, for example, improved pharmacokinetic properties [see,e.g., EP-A 0232262]. On the other hand, for some uses it would bedesirable to be able to delete the Fc part after the fusion protein hasbeen expressed, detected and purified.

"Host cell(s)" is a cell which has been transformed or transfected, oris capable of transformation or transfection by an exogenouspolynucleotide sequence.

"Identity," as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, as thecase may be, as determined by comparing the sequences. In the art,"identity" also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. "Identity"can be readily calculated by known methods, including but not limited tothose described in (Computational Molecular Biology, Lesk, A. M., ed.,Oxford University Press, New York, 1988; Biocomputing: Informatics andGenome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of sequence Data, Part I, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991; and Carillo, H., and Lipman, D., SIAM .J Applied Math.,48: 1073 (1988). Methods to determine identity are designed to give thelargest match between the sequences tested. Moreover, methods todetermine identity are codified in publicly available computer programs.Computer program methods to determine identity between two sequencesinclude, but are not limited to, the GCG program package (Devereux, J.,et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, andFASTA (Altschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990). TheBLAST X program is publicly available from NCBI and other sources (BLASTManual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894;Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990). The well knownSmith Waterman algorithm may also be used to determine identity.

Parameters for polypeptide sequence comparison include the following:

1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)

Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl.Acad. Sci. USA. 89:10915-10919 (1992)

Gap Penalty: 12

Gap Length Penalty: 4

A program useful with these parameters is publicly available as the"gap" program from Genetics Computer Group, Madison Wis. Theaforementioned parameters are the default parameters for peptidecomparisons (along with no penalty for end gaps).

Parameters for polynucleotide comparison include the following:

1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)

Comparison matrix: matches=+10, mismatch=0

Gap Penalty: 50

Gap Length Penalty: 3

Available as: The "gap" program from Genetics Computer Group, MadisonWis. These are the default parameters for nucleic acid comparisons.

A preferred meaning for "identity" for polynucleotides and polypeptides,as the case may be, are provided in (1) and (2) below.

(1) Polynucleotide embodiments further include an isolatedpolynucleotide comprising a polynucleotide sequence having at least a50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to the referencesequence of SEQ ID NO:1, wherein said polynucleotide sequence may beidentical to the reference sequence of SEQ ID NO: 1 or may include up toa certain integer number of nucleotide alterations as compared to thereference sequence, wherein said alterations are selected from the groupconsisting of at least one nucleotide deletion, substitution, includingtransition and transversion, or insertion, and wherein said alterationsmay occur at the 5' or 3' terminal positions of the reference nucleotidesequence or anywhere between those terminal positions, interspersedeither individually among the nucleotides in the reference sequence orin one or more contiguous groups within the reference sequence, andwherein said number of nucleotide alterations is determined bymultiplying the total number of nucleotides in SEQ ID NO:1 by theinteger defining the percent identity divided by 100 and thensubtracting that product from said total number of nucleotides in SEQ IDNO:1, or:

    n.sub.n ≦x.sub.n -(x.sub.n ·y),

wherein n_(n) is the number of nucleotide alterations, x_(n) is thetotal number of nucleotides in SEQ ID NO:1, y is 0.50 for 50%, 0.60 for60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for95%, 0.97 for 97% or 1.00 for 100%, and · the symbol for themultiplication operator, and wherein any non-integer product of x_(n)and y is rounded down to the nearest integer prior to subtracting itfrom x_(n). Alterations of a polynucleotide sequence encoding thepolypeptide of SEQ ID NO:2 may create nonsense, missense or frameshiftmutations in this coding sequence and thereby alter the polypeptideencoded by the polynucleotide following such alterations.

By way of example, a polynucleotide sequence of the present inventionmay be identical to the reference sequence of SEQ ID NO: 1, that is itmay be 100% identical, or it may include up to a certain integer numberof nucleic acid alterations as compared to the reference sequence suchthat the percent identity is less than 100% identity. Such alterationsare selected from the group consisting of at least one nucleic aciddeletion, substitution, including transition and transversion, orinsertion, and wherein said alterations may occur at the 5' or 3'terminal positions of the reference polynucleotide sequence or anywherebetween those terminal positions, interspersed either individually amongthe nucleic acids in the reference sequence or in one or more contiguousgroups within the reference sequence. The number of nucleic acidalterations for a given percent identity is determined by multiplyingthe total number of nucleic acids in SEQ ID NO:1 by the integer definingthe percent identity divided by 100 and then subtracting that productfrom said total number of nucleic acids in SEQ ID NO:1, or:

    n.sub.n ≦x.sub.n -(x.sub.n ·y),

wherein n_(n) is the number of nucleic acid alterations, x_(n) is thetotal number of nucleic acids in SEQ ID NO:1, y is, for instance 0.70for 70%, 0.80 for 80%, 0.85 for 85% etc., · is the symbol for themultiplication operator, and wherein any non-integer product of x_(n)and y is rounded down to the nearest integer prior to subtracting itfrom x_(n).

(2) Polypeptide embodiments further include an isolated polypeptidecomprising a polypeptide having at least a 50,60, 70, 80, 85, 90, 95, 97or 100% identity to a polypeptide reference sequence of SEQ ID NO:2,wherein said polypeptide sequence may be identical to the referencesequence of SEQ ID NO: 2 or may include up to a certain integer numberof amino acid alterations as compared to the reference sequence, whereinsaid alterations are selected from the group consisting of at least oneamino acid deletion, substitution, including conservative andnon-conservative substitution, or insertion, and wherein saidalterations may occur at the amino- or carboxy-terminal positions of thereference polypeptide sequence or anywhere between those terminalpositions, interspersed either individually among the amino acids in thereference sequence or in one or more contiguous groups within thereference sequence, and wherein said number of amino acid alterations isdetermined by multiplying the total number of amino acids in SEQ ID NO:2by the integer defining the percent identity divided by 100 and thensubtracting that product from said total number of amino acids in SEQ IDNO:2, or:

    n.sub.a ≦x.sub.a -(x.sub.a ·y),

wherein n_(a) is the number of amino acid alterations, x_(a) is thetotal number of amino acids in SEQ ID NO:2, y is 0.50 for 50%, 0.60 for60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for95%, 0.97 for 97% or 1.00 for 100%, and · is the symbol for themultiplication operator, and wherein any non-integer product of x_(a)and y is rounded down to the nearest integer prior to subtracting itfrom x_(a).

By way of example, a polypeptide sequence of the present invention maybe identical to the reference sequence of SEQ ID NO:2, that is it may be100% identical, or it may include up to a certain integer number ofamino acid alterations as compared to the reference sequence such thatthe percent identity is less than 100% identity. Such alterations areselected from the group consisting of at least one amino acid deletion,substitution, including conservative and non-conservative substitution,or insertion, and wherein said alterations may occur at the amino- orcarboxy-terminal positions of the reference polypeptide sequence oranywhere between those terminal positions, interspersed eitherindividually among the amino acids in the reference sequence or in oneor more contiguous groups within the reference sequence. The number ofamino acid alterations for a given % identity is determined bymultiplying the total number of amino acids in SEQ ID NO:2 by theinteger defining the percent identity divided by 100 and thensubtracting that product from said total number of amino acids in SEQ IDNO:2, or:

    n.sub.a ≦x.sub.a -(x.sub.a ·y),

wherein n_(a) is the number of amino acid alterations, x_(a) is thetotal number of amino acids in SEQ ID NO:2, y is, for instance 0.70 for70%, 0.80 for 80%, 0.85 for 85% etc., and · is the symbol for themultiplication operator, and wherein any non-integer product of x_(a)and y is rounded down to the nearest integer prior to subtracting itfrom x_(a).

"Immunologically equivalent derivative(s)" as used herein encompasses apolypeptide, polynucleotide, or the equivalent of either which when usedin a suitable formulation to raise antibodies in a vertebrate, theantibodies act to interfere with the immediate physical interactionbetween pathogen and mammalian host.

"Immunospecific" means that characteristic of an antibody whereby itpossesses substantially greater affinity for the polypeptides of theinvention or the polynucleotides of the invention than its affinity forother related polypeptides or polynucleotides respectively, particularlythose polypeptides and polynucleotides in the prior art.

"Individual(s)" means a multicellular eukaryote, including, but notlimited to a metazoan, a mammal, an ovid, a bovid, a simian, a primate,and a human.

"Isolated" means altered "by the hand of man" from its natural state,i.e., if it occurs in nature, it has been changed or removed from itsoriginal environment, or both. For example, a polynucleotide or apolypeptide naturally present in a living organism is not "isolated,"but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is "isolated", as the term is employedherein. Moreover, a polynucleotide or polypeptide that is introducedinto an organism by transformation, genetic manipulation or by any otherrecombinant method is "isolated" even if it is still present in saidorganism, which organism may be living or non-living.

"Organism(s)" means a (i) prokaryote, including but not limited to, amember of the genus Streptococcus, Staphylococcus, Bordetella,Corynebacterium, Mycobacterium, Neisseria, Haemophilus, Actinomycetes,Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella,Pasturella, Moraxella, Acinetobacter, Erysipelothrix, Branhamella,Actinobacillus, Streptobacillus, Listeria, Calymmatobacterium, Brucella,Bacillus, Clostridium, Treponema, Escherichia, Salmonella, Kleibsiella,Vibrio, Proteus, Erwinia, Borrelia, Leptospira, Spirillum,Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas, Rickettsia,Chlamydia, Borrelia and Mycoplasma, and further including, but notlimited to, a member of the species or group, Group A Streptococcus,Group B Streptococcus, Group C Streptococcus, Group D Streptococcus,Group G Streptococcus, Streptococcus pneumoniae, Streptococcus pyogenes,Streptococcus agalactiae, Streptococcus faecalis, Streptococcus faecium,Streptococcus durans, Neisseria gonorrheae, Neisseria meningitidis,Staphylococcus aureus, Staphylococcus epidermidis, Corynebacteriumdiptheriae, Gardnerella vaginalis, Mycobacterium tuberculosis,Mycobacterium bovis, Mycobacterium ulcerans, Mycobacterium leprae,Actinomyctes israelii, Listeria monocytogenes, Bordetella pertusis,Bordatella parapertusis, Bordetella bronchiseptica, Escherichia coli,Shigella dysenteriae, Haemophilus influenzae, Haemophilus aegyptius,Haemophilus parainfluenzae, Haemophilus ducreyi, Bordetella, Salmonellatyphi, Citrobacter freundii, Proteus mirabilis, Proteus vulgaris,Yersinia pestis, Kleibsiella pneumoniae, Serratia marcessens, Serratialiquefaciens, Vibrio cholera, Shigella dysenterii, Shigella flexneri,Pseudomonas aeruginosa, Franscisella tularensis, Brucella abortis,Bacillus anthracis, Bacillus cereus, Clostridium perfringens,Clostridium tetani, Clostridium botulinum, Treponema pallidum,Rickettsia rickettsii and Chlamydia trachomitis, (ii) an archaeon,including but not limited to Archaebacter, and (iii) a unicellular orfilamentous eukaryote, including but not limited to, a protozoan, afungus, a member of the genus Saccharomyces, Kluveromyces, or Candida,and a member of the species Saccharomyces ceriviseae, Kluveromyceslactis, or Candida albicans.

"Polynucleotide(s)" generally refers to any polyribonucleotide orpolydeoxyribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. "Polynucleotide(s)" include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions or single-, double- and triple-stranded regions,single- and double-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded, ortriple-stranded regions, or a mixture of single- and double-strandedregions. In addition, "polynucleotide" as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.As used herein, the term "polynucleotide(s)" also includes DNAs or RNAsas described above that contain one or more modified bases. Thus, DNAsor RNAs with backbones modified for stability or for other reasons are"polynucleotide(s)" as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein. It will be appreciated that a great variety ofmodifications have been made to DNA and RNA that serve many usefulpurposes known to those of skill in the art. The term"polynucleotide(s)" as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including, for example, simple and complex cells."Polynucleotide(s)" also embraces short polynucleotides often referredto as oligonucleotide(s).

"Polypeptide(s)" refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds. "Polypeptide(s)" refers to both short chains, commonly referredto as peptides, oligopeptides and oligomers and to longer chainsgenerally referred to as proteins. Polypeptides may contain amino acidsother than the 20 gene encoded amino acids. "Polypeptide(s)" includethose modified either by natural processes, such as processing and otherpost-translational modifications, but also by chemical modificationtechniques. Such modifications are well described in basic texts and inmore detailed monographs, as well as in a voluminous researchliterature, and they are well known to those of skill in the art. Itwill be appreciated that the same type of modification may be present inthe same or varying degree at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains, and the amino or carboxyl termini.Modifications include, for example, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, GPI anchor formation, hydroxylation,lodination, methylation, myristoylation, oxidation, proteolyticprocessing, phosphorylation prenylation, racemization, glycosylation,lipid attachment, sulfation, gamma-carboxylation of glutamic acidresidues, hydroxylation and ADP-ribosylation, selenoylation, sulfation,transfer-RNA mediated addition of amino acids to proteins, such asarginylation, and ubiquitination. See, for instance, PROTEINS--STRUCTUREAND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman andCompany, New York (1993) and Wold, F., Posttranslational ProteinModifications: Perspectives and Prospects, pgs. 1-12 inPOSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed.,Academic Press, New York (1983); Seifter et al., Meth. Enzymol.182:626-646 (1990) and Rattan et al., Protein Synthesis:Posttranslational Modifications and Aging, Ann. N. Y. Acad. Sci. 663:48-62 (1992). Polypeptides may be branched or cyclic, with or withoutbranching. Cyclic, branched and branched circular polypeptides mayresult from post-translational natural processes and may be made byentirely synthetic methods, as well.

"Recombinant expression system(s)" refers to expression systems orportions thereof or polynucleotides of the invention introduced ortransformed into a host cell or host cell lysate for the production ofthe polynucleotides and polypeptides of the invention.

"Subtraction set" is one or more, but preferably less than 100,polynucleotides comprising at least one polynucleotide of the invention

"Variant(s)" as the term is used herein, is a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusion proteins and truncations inthe polypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. The present invention also includes include variants of each ofthe polypeptides of the invention, that is polypeptides that vary fromthe referents by conservative amino acid substitutions, whereby aresidue is substituted by another with like characteristics. Typicalsuch substitutions are among Ala, Val, Leu and lie; among Ser and Thr;among the acidic residues Asp and Glu; among Asn and Gln; and among thebasic residues Lys and Arg; or aromatic residues Phe and Tyr.Particularly preferred are variants in which several, 5-10, 1-5, 1-3,1-2 or 1 amino acids are substituted, deleted, or added in anycombination. A variant of a polynucleotide or polypeptide may be anaturally occurring such as an allelic variant, or it may be a variantthat is not known to occur naturally. Non-naturally occurring variantsof polynucleotides and polypeptides may be made by mutagenesistechniques, by direct synthesis, and by other recombinant methods knownto skilled artisans.

EXAMPLES

The examples below are carried out using standard techniques, which arewell known and routine to those of skill in the art, except whereotherwise described in detail. The examples are illustrative, but do notlimit the invention.

Example 1

Strain selection, Library Production and Sequencing

The polynucleotide having a DNA sequence given in Table 1 [SEQ ID NO: 1or 3] was obtained from a library of clones of chromosomal DNA ofStreptococcus pneumoniae in E. coli. The sequencing data from two ormore clones containing overlapping Streptococcus pneumoniae DNAs wasused to construct the contiguous DNA sequence in SEQ ID NO: 1. Librariesmay be prepared by routine methods, for example:

Methods 1 and 2 below.

Total cellular DNA is isolated from Streptococcus pneumoniae 0100993according to standard procedures and size-fractionated by either of twomethods.

Method 1

Total cellular DNA is mechanically sheared by passage through a needlein order to size-fractionate according to standard procedures. DNAfragments of up to 11 kbp in size are rendered blunt by treatment withexonuclease and DNA polymerase, and EcoRI linkers added. Fragments areligated into the vector Lambda ZapII that has been cut with EcoRI, thelibrary packaged by standard procedures and E. coli infected with thepackaged library. The library is amplified by standard procedures.

Method 2

Total cellular DNA is partially hydrolyzed with a one or a combinationof restriction enzymes appropriate to generate a series of fragments forcloning into library vectors (e.g., RsaI, Pall, AluI, Bshl2351), andsuch fragments are size-fractionated according to standard procedures.EcoRI linkers are ligated to the DNA and the fragments then ligated intothe vector Lambda ZapII that have been cut with EcoRI, the librarypackaged by standard procedures, and E. coli infected with the packagedlibrary. The library is amplified by standard procedures.

Example 2

The determination of expression during infection of a gene fromStreptococcus pneumoniae

Excised lungs from a 48 hour respiratory tract infection ofStreptococcus pneumoniae 0100993 in the mouse is efficiently disruptedand processed in the presence of chaotropic agents and RNAase inhibitorto provide a mixture of animal and bacterial RNA. The optimal conditionsfor disruption and processing to give stable preparations and highyields of bacterial RNA are followed by the use of hybridisation to aradiolabelled oligonucleotide specific to Streptococcus pneumoniae 16SRNA on Northern blots. The RNAase free, DNAase free, DNA and proteinfree preparations of RNA obtained are suitable for Reverse TranscriptionPCR (RT-PCR) using unique primer pairs designed from the sequence ofeach gene of Streptococcus pneumoniae 0100993.

a) Isolation of tissue infected with Streptococcus pneumoniae 0100993from a mouse animal model of infection (lungs)

Streptococcus pneumoniae 0100993 is grown either on TSA/5% horse bloodplates or in AGCH medium overnight, 37° C., 5% CO₂. Bacteria are thencollected and resuspended in phosphate-buffered saline to an A₆₀₀ ofapproximately 0.4. Mice are anaesthetized with isofluorane and 50 ml ofbacterial suspension (approximately 2×10⁵ bacteria) is administeredintranasally using a pipetman. Mice are allowed to recover and have foodand water ad libitum. After 48 hours, the mice are euthanized by carbondioxide overdose, and lungs are aseptically removed and snap-frozen inliquid nitrogen.

b) Isolation of Streptococcus pneumoniae 0100993 RNA from infectedtissue samples

Infected tissue samples, in 2-ml cryo-strorage tubes, are removed from-80° C. storage into a dry ice ethanol bath. In a microbiological safetycabinet the samples are disrupted up to eight at a time while theremaining samples are kept frozen in the dry ice ethanol bath. Todisrupt the bacteria within the tissue sample, 50-100 mg of the tissueis transfered to a FastRNA tube containing a silica/ceramic matrix(BIO101). Immediately, 1 ml of extraction reagents (FastRNA reagents,BIO101) are added to give a sample to reagent volume ratio ofapproximately 1 to 20. The tubes are shaken in a reciprocating shaker(FastPrep FP120, BIO101) at 6000 rpm for 20-120 sec. The crude RNApreparation is extracted with chloroform/isoamyl alcohol, andprecipitated with DEPC-treated/Isopropanol Precipitation Solution(BIO101). RNA preparations are stored in this isopropanol solution at-80° C. if necessary. The RNA is pelleted (12,000 g for 10 min.), washedwith 75% ethanol (v/v in DEPC-treated water), air-dried for 5-10 min,and resuspended in 0.1 ml of DEPC-treated water, followed by 5-10minutes at 55° C. Finally, after at least 1 minute on ice, 200 units ofRnasin (Promega) is added.

RNA preparations are stored at -80° C. for up to one month. For longerterm storage the RNA precipitate can be stored at the wash stage of theprotocol in 75% ethanol for at least one year at -20° C.

Quality of the RNA isolated is assessed by running samples on 1% agarosegels. 1 x TBE gels stained with ethidium bromide are used to visualisetotal RNA yields. To demonstrate the isolation of bacterial RNA from theinfected tissue 1× MOPS, 2.2M formaldehyde gels are run and vacuumblotted to Hybond-N (Amersham). The blot is then hybridised with a ³²P-labelled oligonucletide probe, of sequence 5'AACTGAGACTGGCTTTAAGAGATTA3' [SEQ ID NO 9], specific to 16S rRNA of Streptococcus pneumoniae. Thesize of the hybridising band is compared to that of control RNA isolatedfrom in vitro grown Streptococcus pneumoniae 0100993 in the Northernblot. Correct sized bacterial 16S rRNA bands can be detected in totalRNA samples which show degradation of the mammalian RNA when visualisedon TBE gels.

c) The removal of DNA from Streptococcus pneumoniae-derived RNA

DNA was removed from 50 microgram samples of RNA by a 30 minutetreatment at 37° C. with 20 units of RNAase-free DNAaseI (GenHunter) inthe buffer supplied in a final volume of 57 microliters.

The DNAase was inactivated and removed by treatment with TRizol LSReagent (Gibco BRL, Life Technologies) according to the manufacturersprotocol. DNAase treated RNA was resuspended in 100 microlitres of DEPCtreated water with the addition of Rnasin as described before.

d) The preparation of cDNA from RNA samples derived from infected tissue

3 microgram samples of DNAase treated RNA are reverse transcribed usinga SuperScript Preamplification System for First Strand cDNA Synthesiskit (Gibco BRL, Life Technologies) according to the manufacturersinstructions. 150 nanogram of random hexamers is used to prime eachreaction. Controls without the addition of SuperScriptII reversetranscriptase are also run. Both +/-RT samples are treated with RNaseHbefore proceeding to the PCR reaction

e) The use of PCR to determine the presence of a bacterial cDNA speciesPCR reactions are set up on ice in 0.2 ml tubes by adding the followingcomponents: 43 microlitres PCR Master Mix (Advanced BiotechnologiesLtd.); 1 microlitre PCR primers (optimally 18-25 basepairs in length anddesigned to possess similar annealing temperatures), each primer at 10mM initial concentration; and 5 microlitres cDNA.

PCR reactions are run on a Perkin Elmer GeneAmp PCR System 9600 asfollows: 2 minutes at 94° C., then 50 cycles of 30 seconds each at 94°C., 50° C. and 72° C. followed by 7 minutes at 72° C. and then a holdtemperature of 20° C. (the number of cycles is optimally 30-50 todetermine the appearance or lack of a PCR product and optimally 8-30cycles if an estimation of the starting quantity of cDNA from the RTreaction is to be made); 10 microlitre aliquots are then run out on 1%1× TBE gels stained with ethidium bromide, with PCR product, if present,sizes estimated by comparison to a 100 bp DNA Ladder (Gibco BRL, LifeTechnologies). Alternatively if the PCR products are convenientlylabelled by the use of a labelled PCR primer (e.g. labelled at the 5'end with a dye) a suitable aliquot of the PCR product is run out on apolyacrylamide sequencing gel and its presence and quantity detectedusing a suitable gel scanning system (e.g. ABI Prism™ 377 Sequencerusing GeneScane software as supplied by Perkin Elmer).

RT/PCR controls may include +/- reverse transcriptase reactions, 16SrRNA primers or DNA specific primer pairs designed to produce PCRproducts from non-transcribed Streptococcus pneumoniae 0100993 genomicsequences.

To test the efficiency of the primer pairs they are used in DNA PCR withStreptococcus pneumoniae 0100993 total DNA. PCR reactions are set up andrun as described above using approx. 1 microgram of DNA in place of thecDNA.

Primer pairs which fail to give the predicted sized product in eitherDNA PCR or RT/PCR are PCR failures and as such are uninformative. Ofthose which give the correct size product with DNA PCR two classes aredistinguished in RT/PCR: 1.Genes which are not transcribed in vivoreproducibly fail to give a product in RT/PCR; and 2.Genes which aretranscribed in vivo reproducibly give the correct size product in RT/PCRand show a stronger signal in the +RT samples than the signal (if at allpresent) in -RT controls Based on these analyses it was discovered thatthis Streptococcus pneumoniae response regulator gene was transcribed invivo.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 9                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1172 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - ATGGGAAAGA CAATTTTACT CGTTGACGAC GAGGTAGAAA TCACAGATAT TC -            #ATCAGAGA     60                                                                 - - TGCTTAATTC AGGCAGGTTA TCAGGTATTG GTAGCCCAAG ATGGACTGGA AG -            #CGATAGAG    120                                                                 - - ATGTTCAAGG AAAAACCGAT TGATTTGATT ATCACAGATG TCATGATGCC TC -            #GGATGGAT    180                                                                 - - GGTTATGATT TAATCAGTGA GGTTCAATAC TTATCACCAG GGCAGCCTTT CC -            #TATTTATT    240                                                                 - - ACTGCTAAGA CCAGTGAACA GGACAAGATT TACGGCCTGA GCTTGGGAGC AG -            #ATGAATTT    300                                                                 - - ATTGCTAAGC CTTTTAGCCC ACGTGAGCTG GTTTTGCGTG TCCACAATAT TT -            #TGCGCCGC    360                                                                 - - CTTCATCGTG GGGGCGAAAC AGAGCTGATT TCCCTTGGCA ATCTAAAAAT GA -            #ATCATAGT    420                                                                 - - AGTCATGAAG TTCAAATAGG AGAAAAAATG CTGGATTTAA CTGTTAAATC AT -            #TTGAATTG    480                                                                 - - CTGTGGATTT TAGCTAGCAA TCCAGAGCGA GTTTTCTCCA AGACAGACCT CT -            #ATGAAAAG    540                                                                 - - ATCTGGAAAG AAGACTACGT GGATGACACC AATACCTTGA ATGTGCATAT CC -            #ATGCTCTT    600                                                                 - - CGACAGGAGC TGGCAAAATA TAGTAGTGAC CAAACGCCCA CTATTAAGAC AG -            #TTTGGGGG    660                                                                 - - TTGGGATATA AGATAGAGAA ACCGAGAGGA CAAACATGAA ACTAAAAAGT TA -            #TATTTTGG    720                                                                 - - TTGGATATAT TATTTCAACC CTCTTAACCA TTTTGGTTGT TTTTTGGGCT GT -            #TCAAAAAA    780                                                                 - - TGCTGATTGC GAAAGGCGAG ATTTACTTTT TGCTTGGGAT GACCATCGTT GC -            #CAGCCTTG    840                                                                 - - TCGGTGCTGG GATTAGTCTC TTTCTCCTAT TGCCAGTCTT TACGTCGTTG GG -            #CAAACTCA    900                                                                 - - AGGAGCATGC CAAGCGGGTA GCGGCCAAGG ATTTTCCTTC AAATTTGGAG GT -            #TCAAGGTC    960                                                                 - - CTGTAGAATT TCAGCAATTA GGGCAAACTT TTAATGAGAT GTCCCATGAT TT -            #GCAGGTAA   1020                                                                 - - GCTTTGATTC CTTGGAAGAA AGCGAACGAG AAAAGGGCTT GATGATTGCC CA -            #GTTGTCGC   1080                                                                 - - ATGATATTAA GACCCCTATC ACTTCGATCC AAGCGACGGT AGAAGGGATT TT -            #GGATGGGA   1140                                                                 - - TTATCAAGGA GTCGGAGCAA GCTCATTACT AC       - #                  - #            1172                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 232 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Met Gly Lys Thr Ile Leu Leu Val Asp Asp Gl - #u Val Glu Ile Thr Asp       1               5  - #                10  - #                15               - - Ile His Gln Arg Cys Leu Ile Gln Ala Gly Ty - #r Gln Val Leu Val Ala                  20      - #            25      - #            30                   - - Gln Asp Gly Leu Glu Ala Ile Glu Met Phe Ly - #s Glu Lys Pro Ile Asp              35          - #        40          - #        45                       - - Leu Ile Ile Thr Asp Val Met Met Pro Arg Me - #t Asp Gly Tyr Asp Leu          50              - #    55              - #    60                           - - Ile Ser Glu Val Gln Tyr Leu Ser Pro Gly Gl - #n Pro Phe Leu Phe Ile      65                  - #70                  - #75                  - #80        - - Thr Ala Lys Thr Ser Glu Gln Asp Lys Ile Ty - #r Gly Leu Ser Leu Gly                      85  - #                90  - #                95               - - Ala Asp Glu Phe Ile Ala Lys Pro Phe Ser Pr - #o Arg Glu Leu Val Leu                  100      - #           105      - #           110                  - - Arg Val His Asn Ile Leu Arg Arg Leu His Ar - #g Gly Gly Glu Thr Glu              115          - #       120          - #       125                      - - Leu Ile Ser Leu Gly Asn Leu Lys Met Asn Hi - #s Ser Ser His Glu Val          130              - #   135              - #   140                          - - Gln Ile Gly Glu Lys Met Leu Asp Leu Thr Va - #l Lys Ser Phe Glu Leu      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Leu Trp Ile Leu Ala Ser Asn Pro Glu Arg Va - #l Phe Ser Lys Thr        Asp                                                                                             165  - #               170  - #               175             - - Leu Tyr Glu Lys Ile Trp Lys Glu Asp Tyr Va - #l Asp Asp Thr Asn Thr                  180      - #           185      - #           190                  - - Leu Asn Val His Ile His Ala Leu Arg Gln Gl - #u Leu Ala Lys Tyr Ser              195          - #       200          - #       205                      - - Ser Asp Gln Thr Pro Thr Ile Lys Thr Val Tr - #p Gly Leu Gly Tyr Lys          210              - #   215              - #   220                          - - Ile Glu Lys Pro Arg Gly Gln Thr                                          225                 2 - #30                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1100 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - GCAGGTTATC AGGTATTGGT AGCCCAAGAT GGACTGGAAG CGATAGAGAT GT -             #TCAAGGAA     60                                                                 - - AAACCGATTG ATTTGATTAT CACAGATGTC ATGATGCCTC GGATGGATGG TT -            #ATGATTTA    120                                                                 - - ATCAGTGAGG TTCAATACTT ATCACCAGGG CAGCCTTTCC TATTTATTAC TG -            #CTAAGACC    180                                                                 - - AGTGAACAGG ACAAGATTTA CGGCCTGAGC TTGGGAGCAG ATGAATTTAT TG -            #CTAAGCCT    240                                                                 - - TTTAGCCCAC GTGAGCTGGT TTTGCGTGTC CACAATATTT TGCGCCGCCT TC -            #ATCGTGGG    300                                                                 - - GGCGAAACAG AGCTGATTTC CCTTGGCAAT CTAAAAATGA ATCATAGTAG TC -            #ATGAAGTT    360                                                                 - - CAAATAGGAG AAAAAATGCT GGATTTAACT GTTAAATCAT TTGAATTGCT GT -            #GGATTTTA    420                                                                 - - GCTAGCAATC CAGAGCGAGT TTTCTCCAAG ACAGACCTCT ATGAAAAGAT CT -            #GGAAAGAA    480                                                                 - - GACTACGTGG ATGACACCAA TACCTTGAAT GTGCATATCC ATGCTCTTCG AC -            #AGGAGCTG    540                                                                 - - GCAAAATATA GTAGTGACCA AACGCCCACT ATTAAGACAG TTTGGGGGTT GG -            #GATATAAG    600                                                                 - - ATAGAGAAAC CGAGAGGACA AACATGAAAC TAAAAAGTTA TATTTTGGTT GG -            #ATATATTA    660                                                                 - - TTTCAACCCT CTTAACCATT TTGGTTGTTT TTTGGGCTGT TCAAAAAATG CT -            #GATTGCGA    720                                                                 - - AAGGCGAGAT TTACTTTTTG CTTGGGATGA CCATCGTTGC CAGCCTTGTC GG -            #TGCTGGGA    780                                                                 - - TTAGTCTCTT TCTCCTATTG CCAGTCTTTA CGTCGTTGGG CAAACTCAAG GA -            #GCATGCCA    840                                                                 - - AGCGGGTAGC GGCCAAGGAT TTTCCTTCAA ATTTGGAGGT TCAAGGTCCT GT -            #AGAATTTC    900                                                                 - - AGCAATTAGG GCAAACTTTT AATGAGATGT CCCATGATTT GCAGGTAAGC TT -            #TGATTCCT    960                                                                 - - TGGAAGAAAG CGAACGAGAA AAGGGCTTGA TGATTGCCCA GTTGTCGCAT GA -            #TATTAAGA   1020                                                                 - - CCCCTATCAC TTCGATCCAA GCGACGGTAG AAGGGATTTT GGATGGGATT AT -            #CAAGGAGT   1080                                                                 - - CGGAGCAAGC TCATTACTAC            - #                  - #                     110 - #0                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 208 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Ala Gly Tyr Gln Val Leu Val Ala Gln Asp Gl - #y Leu Glu Ala Ile Glu       1               5  - #                10  - #                15               - - Met Phe Lys Glu Lys Pro Ile Asp Leu Ile Il - #e Thr Asp Val Met Met                  20      - #            25      - #            30                   - - Pro Arg Met Asp Gly Tyr Asp Leu Ile Ser Gl - #u Val Gln Tyr Leu Ser              35          - #        40          - #        45                       - - Pro Gly Gln Pro Phe Leu Phe Ile Thr Ala Ly - #s Thr Ser Glu Gln Asp          50              - #    55              - #    60                           - - Lys Ile Tyr Gly Leu Ser Leu Gly Ala Asp Gl - #u Phe Ile Ala Lys Pro      65                  - #70                  - #75                  - #80        - - Phe Ser Pro Arg Glu Leu Val Leu Arg Val Hi - #s Asn Ile Leu Arg Arg                      85  - #                90  - #                95               - - Leu His Arg Gly Gly Glu Thr Glu Leu Ile Se - #r Leu Gly Asn Leu Lys                  100      - #           105      - #           110                  - - Met Asn His Ser Ser His Glu Val Gln Ile Gl - #y Glu Lys Met Leu Asp              115          - #       120          - #       125                      - - Leu Thr Val Lys Ser Phe Glu Leu Leu Trp Il - #e Leu Ala Ser Asn Pro          130              - #   135              - #   140                          - - Glu Arg Val Phe Ser Lys Thr Asp Leu Tyr Gl - #u Lys Ile Trp Lys Glu      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Asp Tyr Val Asp Asp Thr Asn Thr Leu Asn Va - #l His Ile His Ala        Leu                                                                                             165  - #               170  - #               175             - - Arg Gln Glu Leu Ala Lys Tyr Ser Ser Asp Gl - #n Thr Pro Thr Ile Lys                  180      - #           185      - #           190                  - - Thr Val Trp Gly Leu Gly Tyr Lys Ile Glu Ly - #s Pro Arg Gly Gln Thr              195          - #       200          - #       205                      - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1097 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - AGATAGAGAA ACCGAGAGGA CAAACATGAA ACTAAAAAGT TATATTTTGG TT -             #GGATATAT     60                                                                 - - TATTTCAACC CTCTTAACCA TTTTGGTTGT TTTTTGGGCT GTTCAAAAAA TG -            #CTGATTGC    120                                                                 - - GAAAGGCGAG ATTTACTTTT TGCTTGGGAT GACCATCGTT GCCAGCCTTG TC -            #GGTGCTGG    180                                                                 - - GATTAGTCTC TTTCTCCTAT TGCCAGTCTT TACGTCGTTG GGCAAACTCA AG -            #GAGCATGC    240                                                                 - - CAAGCGGGTA GCGGCCAAGG ATTTTCCTTC AAATTTGGAG GTTCAAGGTC CT -            #GTAGAATT    300                                                                 - - TCAGCAATTA GGGCAAACTT TTAATGAGAT GTCCCATGAT TTGCAGGTAA GC -            #TTTGATTC    360                                                                 - - CTTGGAAGAA AGCGAACGAG AAAAGGGCTT GATGATTGCC CAGTTGTCGC AT -            #GATATTAA    420                                                                 - - GACCCCTATC ACTTCGATCC AAGCGACGGT AGAAGGGATT TTGGATGGGA TT -            #ATCAAGGA    480                                                                 - - GTCGGAGCAA GCTCATTATC TAGCAACCAT TGGACGCCAG ACGGAGAGGC TC -            #AATAAACT    540                                                                 - - GGTTGAGGAG TTGAATTTTT TGACCCTAAA CACAGCTAGA AATCAGGTGG AA -            #ACTACCAG    600                                                                 - - TAAAGACAGT ATTTTTCTGG ACAAGCTCTT AATTGAGTGC ATGAGTGAAT TT -            #CAGTTTTT    660                                                                 - - GATTGAGCAG GAGAGAAGAG ATGTCCACTT GCAGGTAATC CCAGAGTCTG CC -            #CGGATTGA    720                                                                 - - GGGAGATTAT GCTAAGCTTT CTCGTATCTT GGTGAATCTG GTCGATAACG CT -            #TTTAAATA    780                                                                 - - TTCTGCTCCA GGAACCAAGC TGGAAGTGGT GACTAAGCTG GAGAAGGGCC AG -            #CTTTCAAT    840                                                                 - - CAGTGTGACC GATGAAGGGC AGGGCATTGC CCCAGAGGAT TTGGAAAATA TT -            #TTCAAACG    900                                                                 - - CCTTTATCGT GTCGAAACTT CGCGTAACAT GAAGACAGGT GGTCATGGAT TA -            #GGACTTGC    960                                                                 - - GATTGCGCGT GAATTGGCCC ATCAATTGGG TGGGGAAATC ACAGTCAGCA GC -            #CAGTACGG   1020                                                                 - - TCTAGGAAGT ACCTTTACCC TCGTTCTCAA TCTCTCTGGT AGTGAAAATA AA -            #GCCTAAAA   1080                                                                 - - CCCCTTTACA AATCCAG             - #                  - #                      - # 1097                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 350 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - Met Lys Leu Lys Ser Tyr Ile Leu Val Gly Ty - #r Ile Ile Ser Thr Leu       1               5  - #                10  - #                15               - - Leu Thr Ile Leu Val Val Phe Trp Ala Val Gl - #n Lys Met Leu Ile Ala                  20      - #            25      - #            30                   - - Lys Gly Glu Ile Tyr Phe Leu Leu Gly Met Th - #r Ile Val Ala Ser Leu              35          - #        40          - #        45                       - - Val Gly Ala Gly Ile Ser Leu Phe Leu Leu Le - #u Pro Val Phe Thr Ser          50              - #    55              - #    60                           - - Leu Gly Lys Leu Lys Glu His Ala Lys Arg Va - #l Ala Ala Lys Asp Phe      65                  - #70                  - #75                  - #80        - - Pro Ser Asn Leu Glu Val Gln Gly Pro Val Gl - #u Phe Gln Gln Leu Gly                      85  - #                90  - #                95               - - Gln Thr Phe Asn Glu Met Ser His Asp Leu Gl - #n Val Ser Phe Asp Ser                  100      - #           105      - #           110                  - - Leu Glu Glu Ser Glu Arg Glu Lys Gly Leu Me - #t Ile Ala Gln Leu Ser              115          - #       120          - #       125                      - - His Asp Ile Lys Thr Pro Ile Thr Ser Ile Gl - #n Ala Thr Val Glu Gly          130              - #   135              - #   140                          - - Ile Leu Asp Gly Ile Ile Lys Glu Ser Glu Gl - #n Ala His Tyr Leu Ala      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Thr Ile Gly Arg Gln Thr Glu Arg Leu Asn Ly - #s Leu Val Glu Glu        Leu                                                                                             165  - #               170  - #               175             - - Asn Phe Leu Thr Leu Asn Thr Ala Arg Asn Gl - #n Val Glu Thr Thr Ser                  180      - #           185      - #           190                  - - Lys Asp Ser Ile Phe Leu Asp Lys Leu Leu Il - #e Glu Cys Met Ser Glu              195          - #       200          - #       205                      - - Phe Gln Phe Leu Ile Glu Gln Glu Arg Arg As - #p Val His Leu Gln Val          210              - #   215              - #   220                          - - Ile Pro Glu Ser Ala Arg Ile Glu Gly Asp Ty - #r Ala Lys Leu Ser Arg      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Ile Leu Val Asn Leu Val Asp Asn Ala Phe Ly - #s Tyr Ser Ala Pro        Gly                                                                                             245  - #               250  - #               255             - - Thr Lys Leu Glu Val Val Thr Lys Leu Glu Ly - #s Gly Gln Leu Ser Ile                  260      - #           265      - #           270                  - - Ser Val Thr Asp Glu Gly Gln Gly Ile Ala Pr - #o Glu Asp Leu Glu Asn              275          - #       280          - #       285                      - - Ile Phe Lys Arg Leu Tyr Arg Val Glu Thr Se - #r Arg Asn Met Lys Thr          290              - #   295              - #   300                          - - Gly Gly His Gly Leu Gly Leu Ala Ile Ala Ar - #g Glu Leu Ala His Gln      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Leu Gly Gly Glu Ile Thr Val Ser Ser Gln Ty - #r Gly Leu Gly Ser        Thr                                                                                             325  - #               330  - #               335             - - Phe Thr Leu Val Leu Asn Leu Ser Gly Ser Gl - #u Asn Lys Ala                          340      - #           345      - #           350                  - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - ATGGGAAAGA CAATTTTACT CGTTG          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - TGTTTGTCCT CTCGGTTTCT CTATC          - #                  - #                   25                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - AACTGAGACT GGCTTTAAGA GATTA          - #                  - #                   25                                                                    __________________________________________________________________________

What is claimed is:
 1. An isolated protein comprising a polypeptidecomprising the amino acid sequence set forth in SEQ ID NO:2.
 2. Acomposition comprising the isolated protein of claim 1 and apharmaceutically acceptable carrier.
 3. An isolated fusion proteincomprising a heterologous amino acid sequence fused to the polypeptideof claim
 1. 4. A composition comprising the isolated fusion protein ofclaim 3 and a pharmaceutically acceptable carrier.
 5. The isolatedprotein of claim 1, wherein the isolated protein consists of the aminoacid sequence set forth in SEQ ID NO:2.
 6. A composition comprising theisolated protein of claim 5 and a pharmaceutically acceptable carrier.7. An isolated protein comprising a polypeptide comprising the aminoacid sequence set forth in SEQ ID NO:4.
 8. An isolated fusion proteincomprising a heterologous amino acid sequence fused to the polypeptideof claim
 7. 9. The isolated protein of claim 7, wherein the isolatedprotein consists of the amino acid sequence set forth in SEQ ID NO:4.10. An isolated protein comprising a polypeptide comprising at least 50consecutive amino acids of SEQ ID NO:2.
 11. An isolated fusion proteincomprising a heterologous amino acid sequence fused to the polypeptideof claim
 10. 12. An isolated protein comprising a polypeptide comprisingat least 30 consecutive amino acids of SEQ ID NO:2.
 13. An isolatedfusion protein comprising a heterologous amino acid sequence fused tothe polypeptide of claim
 12. 14. A process for diagnosing aStreptococcal infection in an individual comprising the stepof:analyzing for the presence of the polypeptide of claim 1 in a samplederived from said individual.